Borna disease viral sequences, diagnostics and therapeutics for nervous system diseases

ABSTRACT

The present invention presents: genomic nucleotide sequence of Borna disease virus, nucleotide and amino acid sequences of Borna disease virus proteins, recombinant viral proteins, vectors and cells containing the sequences or encoding the proteins, ligand binding to these proteins such as antibodies, and the diagnostic and therapeutic uses of the foregoing.

This invention was made with Government support under Grant No.NS-29425, awarded by the National Institutes of Health. The Governmenthas certain rights in this invention.

This patent application is a continuation-in-part of U.S. patentapplication Ser. No. 08/369,822, filed on Jan. 6, 1995, now U.S. Pat.No. 6,015,660.

FIELD OF THE INVENTION

The present invention relates to the field of virology, immunology, genetherapy, transplantation of viral transfected cells, and in vivochemical delivery.

BACKGROUND OF THE INVENTION

Borna disease is an immune-mediated neurologic syndrome {Narayan, O., etal., Science 220:1401-1403 (1983)} caused by infection with Bornadisease virus (BDV). BDV is a neurotropic, nonsegmented andnegative-strand RNA virus that causes a progressive, immune-mediatedneurologic disease characterized by disturbances in movement andbehavior {Ludwig, H., et al., Prog. Med. Virol, 35:107-151). It causesfatal disease in expensive domestic animals. Although natural infectionwas originally considered to be restricted to horses and sheep inSoutheastern Germany, recent studies suggest that BDV infects horses inNorth America {Kao, M., et al., Vet. Rec., 132:241-4 (1993)}, cats inSweden {Lundgren, A.-L., et al., Zbl. Vet. Med. [B], 40:298-303 (1993)},ostriches in Israel {Malkinson, M., et al., Vet. Rec., 133:304 (1993)}and some human subjects with neuropsychiatric disorders in Europe andNorth America {Bode, L., et al., Arch. Virol. [Suppl], 7:159-167 (1993);Bode, L., et al., Lancet, ii:689 (1988); Fu, Z. F., et al., J. Affect.Disorders, 27:61-68 (1993) and Rott, R., et al., Science, 228:755-756(1985)}.

Experimental infection in rats {Narayan, O., et al., Science,220:1401-1403 (1983)} results in a multiphasic syndrome characterized byhyperactivity, stereotyped behaviors, dyskinesias and dystonias.

Though natural infection has not been reported in primates, subhumanprimates can be infected experimentally {Sprankel, H., et al., Med.Microbiol. Immunol. 165:1-18 (1978) and Stitz, L., et al., J. Med.Virol. 6:333-340 (1980)}. Antibodies to BDV proteins have been found inpatients with neuropsychiatric disorders (Rott, R., et al., Science228:755-756 (1985); Fu, Z. F., et al., J. Affective Disord. 27:61-68(1993) and Bode, L., et al., Arch. Virol. (Suppl.) 7:159-167 (1993)}.

Because BDV grows only to low titer, it was difficult to purify foranalysis. However, the identification of BDV cDNA clones by subtractivehybridization {Lipkin, W. I., et al., Proc. Natl. Acad. Sci. USA87:4184-4188 (1990) and VandeWoude, S., et al., Science 250:1276-1281(1990)} and, more recently, the advent of a method for isolation ofvirus particles {Briese, T., et al., Proc. Natl. Acad. Sci. USA89:11486-11489 (1992)} led to partial characterization of BDV as anegative-strand RNA virus which transcribes its RNA in the cell nucleus{Briese, T., et al., Proc. Natl. Acad. Sci. USA 89:11486-11489 (1992)}.

The diagnosis of BDV infection is based on the appearance of a clinicalsyndrome consistent with disease, and the presence of serum antibodiesthat detect viral proteins in infected cells by indirectimmnunofluorescent test (IFT) {Pauli, G., et al., Zbl. Vet. Med. [B]31:552-557 (1984)}, Western blot (WB; immunoblotting) orimmunoprecipitation (IP) {Ludwig, H., et al., Prog. Med. Virol,35:107-151 (1988)}. These methods are cumbersome and difficult to usefor large surveys of human and livestock populations.

SUMMARY OF THE INVENTION

One aspect of the invention presents the nucleotide and amino acidsequences of Borna disease virus (BDV), their derivatives, the vectorsfor expressing them, and cells transfected by these vectors.

Another aspect of the invention presents novel BDV viral proteins gp18and p57 and their respective recombinant proteins, recp18 and recp57.Also disclosed are their nucleotide and amino acid sequences, vectorsencoding them, cells transfected by these vectors, and antibodiesdirected to these proteins.

Another aspect of the invention presents assays for detecting ligandswhich bind BDV proteins or their derivatives. Preferably, these assaysare immunoassays for detecting antibodies to BDV protein or itsderivatives. The assays are useful for detecting: (1) BDV infection orrelated pathogenesis; and (2) neurologic and neuropsychiatric diseasenot due to BDV infection. Preferably, p40, p23 or gp18, and theirsynthetic versions or fragments are used in these assays. The preferredimmunoassays are enzyme-linked immunosorbent assays (ELISAs) based onthe use of recombinant viral proteins: recp40, recp23, and/or recp18,and/or the immunoreactive fragments of the foregoing, to detect ligands,such as antibodies, in the patient's biological sample, that areimmunoreactive with these proteins. The assay can also be used tomonitor the diseases by monitoring the titer of such ligands. The titerof the ligands can also be prognosticative of the diseases.

Another aspect of the invention presents alternative methods fordetecting the above diseases by detecting the hybridization ofnucleotide sequences in a patient's biological sample with thenucleotide sequences coding for BDV protein or its derivatives.

Another aspect of the invention presents assay kits for the abovediagnostic tests.

Another aspect of the invention presents vaccines against the abovediseases.

Another aspect of the invention presents synthetic peptides, based ontruncated BDV protein, useful for immunoassays for detecting antibodiesto BDV or for raising antibodies for the therapeutic uses described inthe next paragraph. The method for obtaining these peptides are alsopresented.

Another aspect of the invention presents methods, using ligands orchemicals such as antibodies, capable of binding to BDV proteins ortheir derivatives, for treating: (1) BDV infection or relatedpathogenesis; and (2) neurologic and neuropsychiatric disease not due toBDV infection. Examples of such antibodies are those specific to gp18and p57. Also presented are these therapeutic agents, methods forscreening for them, especially those that bind to the immunogenicepitopes of BDV protein. The methods for producing the antibodies arealso presented.

Another aspect of the invention presents a BDV-based viral vector usefulfor in vivo delivery of genes and chemicals to the nervous system. Alsodisclosed are: the cells transfected by the viral vector and cell linesderived therefrom, the in vitro harvesting of the gene product from suchcells and cell lines, and the transplant of such cells into animals.

Other aspects and advantages of the invention will be apparent to thoseskilled in the art upon consideration of the following detaileddescription which provides illustrations of the invention in itspresently preferred embodiments.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 presents the genomic organization and transcriptional map of BDV.

FIG. 2 shows the complete genomic sequence of BDV (strain V) in 5' to 3'cDNA (SEQ ID NO:19) with the deduced amino acid sequence shown below thecDNA.

FIG. 3 (a) presents the organization of the BDV genome; (b) presents thecoding potential of the genome.

FIG. 4 shows alignment of the p180 (also referred to as "pol") openreading frame (ORF) and negative-strand RNA virus L-polymerase aminoacid sequences with PILEUP computer program (Sequence Analysis SoftwarePackage, Genetics Computer, Inc., Madison, Wis.). BDV sequence (aminoacids 377 to 829 of SEQ ID NO:10) is indicated with double arrowheads.Rhabdoviridae: RaV, rabies virus (SEQ ID NO:33); VSV, vesicularstomatitis virus (SEQ ID NO:34); SYN, sonchus yellow net virus (SEQ IDNO:35). Paramyxoviridae: MeV, measles virus (SEQ ID NO:36); SeV, Sendaivirus (SEQ ID NO:37); NDV, Newcastle disease virus (SEQ ID NO:38); RSV,respiratory syncytial virus (SEQ ID NO:40). Filoviridae: MaV, Marburgvirus.

FIG. 5 presents sequence analysis of BDV genomic termini. (a) Similarityof 3'-terminal BDV sequence to Rhabdoviridae: RaV (SEQ ID NO:47) and VSV(SEQ ID NO:48); Paramyxoviridae: MeV (SEQ ID NO:46), SeV (SEQ ID NO:44),NDV (SEQ ID NO:45), and RSV (SEQ ID NO:41); and Filoviridae: MaV (SEQ IDNO:43) and Ebola virus (EboV, SEQ ID NO:42) (b) Comparison ofcomplementarity at 3' and 5' termini of BDV genomic RNA with that offour other nonsegmented, negative-strand RNA viruses: RSV (SEQ ID NO:49and NO:50), MaV (SEQ ID NO:51 and NO:52), SeV (SEQ ID NO:53 and NO:54),and RaV (SEQ ID NO:55 and NO:56).

FIG. 6 presents the map of BDV subgenomic RNAs relative to the viralantigenome. (a) Northern hybridization analysis of rat brain poly(A)⁺RNA; (b) position of viral transcripts with respect to antigenome asdetermined by Northern hybridization and sequence analysis; (c)alignment of the seven potential termination sites of BDV. SEQ ID NO:19positions 1156-1201 (p40/t1/T2); 1856-1901 (p23/T3); 2328-2417 (gp18);3747-3781 (p57/t4); 4474-4520 (T5); 4738-4783 (t6); and 8819-8874(p180/T7).

FIG. 7 presents the sequence of ORF gp18 (SEQ ID NOS 5 and 6).

FIG. 8 shows glycan determination of gp18. Lanes: 0, protein detectionby mouse anti-gp18 serum; 1, ConA; 2, wheat germ agglutinin; 3, D.stramonium agglutinin; 4, BS-I; 5, BS-II; 6, G. nivalis agglutinin; 7,S. nigra agglutinin; 8, M. amrensis agglutinin; 9, peanut agglutinin.Positions of molecular weight markers are shown in kilodaltons at theright.

FIG. 9 presents treatment of gp18 with buffer alone or endoglycosidase.Lanes: 1, buffer; 2, endoglycosidase F plus N-glycosidase F; 3,endoglycosidase F (N-glycosidase free); 4, endo-β-galactosidase.Positions of molecular weight markers are shown in kilodaltons at theright.

FIG. 10 presents in vitro transcription, translation, andcotranslational processing of gp18. (A) Lanes: 1, pBDV-23 RNA; 2,pBDV-23 RNA plus microsomal membranes; 3, pBDV-gp18 RNA; 4, pBDV-gp18RNA plus microsomal membranes; 5, pBDV-gp18 RNA plus microsomalmembranes, incubated with endoglycosidases. (B) Lanes: 1, pBDV-gp18 RNA;2, pBDV-gp18 RNA plus microsomal membranes; 3, pBDV-gp18 RNA plusmicrosomal membranes, incubated with endoglycosidases.

FIG. 11 presents Western blot analysis of native and recombinantproteins with monospecific antisera to recombinant proteins and serafrom infected rats. (A) Lane 1, C6BDV lysate; lane 2, recp40; lane 3,recp23; lane 4, recp18; lane 5, C6 lysate; lane 6, recp40, recp23 andrecp18. Lanes 1-4 were treated with serum from infected rat; lanes 5 and6 were treated with serum from noninfected rat. (B) C6BDV lysates (lanes1-3) and C6 lysates (lanes 4 and 5) were incubated with: lanes 1 and 4,serum from infected rat; lane 2, anti-p40 rabbit serum; lane 3, anti-p23rabbit serum; and lane 5, pooled anti-p40 and anti-p23 sera.

FIG. 12 presents ELISA of infected rat serum reacted with recp40.Circles, recp40 and serum from chronically infected rat; squares, recp40and serum from noninfected rat; triangles, BSA and serum fromchronically infected rat.

FIG. 13 presents timecourse for the appearance of antibodies toBDV-proteins. (A) recp40; (B) recp23; and (C) recp18.

FIG. 14 presents timecourse for the appearance of antibodies to BDVproteins in sera from individual rats after intranasal infection. (A)Neutralization activity in sera from BDV-infected rats at threetimepoints (5, 10 and 15 weeks post-infection). (B) Plot of mean recp18ELISA titers (open columns) with neutralization titers (hatched columns)at three time points (5, 10 and 15 weeks post-infection). Sera analyzedwere the same as those in panel A. (C) Timecourse for the appearance ofantibodies to recp40, recp23, and gp18 by Western blot analysis.

FIG. 15 presents (A) Immunoprecipitation of gp18 with monoclonalantibodies (Mabs). Lanes: 1, serum from infected rat (15 week pi); 2,serum from noninfected rat; 3, MAb 14/29A5; 4, MAb 14/26B9; 5, MAb14/8E1; 6, MAb 14/13E10; 7, MAb 14/18H7; 8, MAb 24/36F1 (MAb directedagainst the BDV 23 kDa protein, negative control); 9, no antibody. (B)MAbs were analyzed for binding to native gp18 in Western blot. Lanes: 1,serum from infected rat (15 week p.i., D2); 2, serum from noninfectedrat; 3, MAb 14/29A5; 4, MAb 14/26B9; 5, MAb 14/8E1; 6, MAb 14/13E10; 7,MAb 14/18H7; and 8, MAb 24/36F1 (MAb directed against the BDV 23 kDaprotein, negative control).

FIG. 16 presents neutralization profile of sera and MAbs. (A) Serum fromnoninfected rat. (B) serum from infected rat (15 week p.i., D2). (C) MAb14/13E10. (D) MAb 14/29A5.

FIG. 17 presents precipitation of BDV with sera from infected rats. (A)Lanes: 1, serum from infected rat, 15 week p.i.; 2, serum from infectedrat, 5 week p.i.; 3, serum from infected rat, 15 week p.i., no BDV; 4,serum from infected rat, 15 week p.i., genome sense primer used forfirst strand cDNA synthesis. (B) Precipitation of BDV by monospecificantisera to recp18 and MAbs to gp18. Lanes: 1, monospecific rat antiserato recp18; 2, MAb 14/13E10; 3, MAb 14/29A5. DNA markers (basepairs) areshown at the right.

FIG. 18 presents the cDNA of BDV polymerase (SEQ ID NO:19, positions2393-2409 and 3704-8821). "V" denotes the site of its intron which islocated between nucleotide positions 2410 and 3703 in the figure. "I-2",denotes that this is the second intron in the BDV genome.

FIG. 19 presents the partial cDNA genomic sequence for BDV strain HE/80(SEQ ID NO:58).

FIG. 20 graphically presents in A) the immunoreaction of truncatedrecp23 protein fragments with sera from 7 human schizophrenics (SZHuman), 4 BDV infected horses (BD Horse) and 6 BDV infected rats (BDRat); and in B) the truncated recp23 fragments.

FIG. 21 graphically presents in A) the immunoreaction of truncatedunglycosylated recp18 protein fragments with sera from 7 humanschizophrenics (SZ Human), 6 BDV infected rats (BD Rat) and 2 miceimmunized with native gp18 (Mouse α gp18); and in B) the truncatedunglycosylated recp18 fragments.

FIG. 22 graphically presents the overlapping 8-mer peptides, derivedfrom p23, lined up diagonally from the amino (left) terminus to thecarboxyl (right) terminus. Above the overlapping peptides are blocksindicating the immunoreactive regions of p23 and presenting the mappedepitopes and their sequences.

FIG. 23 graphically presents the overlapping 8-mer peptides, derivedfrom unglycosylated recp18, lined up diagonally from the amino (left)terminus to the carboxyl (right) terminus. Above the overlappingpeptides are blocks indicating the immunoreactive regions ofunglycosylated gp18 and presenting the mapped epitopes and theirsequences.

FIG. 24 graphically presents A) the SPOTs tests; B) the locations ofimmunoepitopes along the length of unglycosylated gp18 which areimmunoreactive with the sera in the SPOTs tests of FIG. 24A. Thesequences of the most immunoreactive epitopes are shown. The scaleindicates by the darkness of the spots, the degree of immunoreaction.The lightest shade (Scale 1) indicates no detectable immunoreactivity;the darkest shade (Scale 4) indicates highest immunoreactivity.

DETAILED DESCRIPTION OF THE INVENTION BDV Protein, its Amino Acid andNucleotide Sequences

Table 1 identifies the sequence ID Nos. with their respective nucleotideand amino acid sequences.

                  TABLE 1                                                         ______________________________________                                        Nucleotide and Amino Acid Sequences of Borna Disease                          Virus (BDV)                                                                   ______________________________________                                        Nucleotide Sequence                                                                           Sequence ID No.                                               ______________________________________                                        p40             1                                                             p23             3                                                             gp 18           5                                                             p57             7                                                             BDV polymerase  9                                                             BDV genomic cDNA                                                                              19                                                            Amino Acid Sequence                                                                           Sequence ID No.                                               ______________________________________                                        p40             2                                                             p23             4                                                             gp 18           6                                                             p57             8                                                             BDV polymerase  10                                                            ______________________________________                                    

BDV polymerase is also referred to as "pol" or "p180".

The present application discloses the complete BDV genomic nucleotidesequence, the locations on the genomic nucleotide sequence which encodethe different BDV proteins, the sites of splicing and overlap (see FIGS.1 and 2). Also disclosed are the novel nucleotide and amino acidsequences of BDV proteins gp18, pol and p57. The following FIGS. 1, 2,19, and Table 1 summarize this information.

FIG. 1 shows the genomic organization and transcriptional map of BDV.The BDV genome is shown as a solid line in 3' to 5' direction. Codingregions and their respective reading frames are represented as boxes atthe top; the number above each upward vertical line indicates thenucleotide position of the first AUG codon in the respective ORF.Transcription initiation sites and their nucleotide positions on theviral genome (BDV strain V) are represented by arrows pointingdownstream. Transcription termination sites and splice sites areindicated by downward vertical lines. Dashed lines indicate thatreadthrough at termination sites T2 and T3 results in synthesis oflonger RNAs terminating at T3 and T4, respectively. The 1.2 kb and 0.8kb RNA have been shown to represent the mRNAs for p40 and p23,respectively. p23 could also be translated from the 3.5 kb RNA.Transcripts that are likely to represent mRNAs for gp18, p57 and pol areindicated. Note that gp18 can only be translated from RNAs containingintron 1. Splicing of intron 1 preserves the gp18 initiation codon butintroduces a stop codon such that only the first 13 amino acids could betranslated from the 2.7 (7.0) kb transcripts and the RNA or the 1.4 kbRNA serve as messages for the translation of BDV proteins.

FIG. 2 shows the complete genomic sequence of BDV (strain V) in 5' to 3'cDNA. The deduced amino acid sequences are shown for p40, p23, gp18, p57and pol. Note: the full amino acid sequence for pol after splicingmodification is shown in sequence ID No. 10. The stars (*) indicate stopcodons. Information on transcription and splicing of the genomicsequence is found in Schneider, P. A. et al., J. Virol., 68:5007-5012(1994) and Schneemann, A., et al., J. Virol., 68:6514-6522 (1994), bothreferences are hereby incorporated by reference in their entirety.

FIG. 19 presents the partial cDNA genomic sequence of BDV strain HE/80.Position 1 to 2651 of this sequence corresponds to position 1397 through4054 of the cDNA genomic sequence of BDV strain V. The cDNA sequence ofBDV strain HE/80 disclosed herein encodes part of the p23 and BDVpolymerase proteins, and the complete gp18 and p57 proteins.

The term "nucleotide sequence" as used herein, unless otherwisemodified, includes both ribonucleic acid (RNA) and deoxyribonucleic acid(DNA).

The sequences in Table 1 include both native and synthetic sequences.Unless otherwise modified, the term "protein" as used herein encompassesboth native and synthetic polypeptide and peptide. Synthetic proteinincludes recombinant and chemically synthesized protein. Unlessotherwise indicated, "gp18", "p57", and "pol" proteins include boththeir native and synthetic versions. "recp18", "recp57" and "recpol" arerecombinant proteins of "gp18", "p57", and "pol" proteins, respectively.

Some of the nucleotide sequences disclosed are in the form of DNA. Forexample, SEQ ID No. 19 presents the BDV viral genomic sequence as cDNAof BDV viral genomic RNA. One skilled in the art would realize that theBDV viral genomic RNA is complementary to its cDNA that is shown in FIG.2. The term "BDV genomic nucleotide sequence" thus includes both thefull cDNA and RNA sequences of the BDV genome. Further, as used in thisapplication and claims, the SEQ ID Nos. and disclosed sequences include:(1) the DNA sequences as disclosed, (2) the complementary nucleotidesequences (which may be RNA or DNA) to the disclosed sequences, (3) thecorresponding RNA sequences to the listed DNA sequences wherein theThymidine ("T") in the disclosed DNA sequences is replaced with Uracil("U"), (4) nucleotide sequences wherein other nucleotides known in theart such as nucleotide analogs, replace those in the foregoingsequences, for example, 5-methyl-cytosine replacing cytosine, and (5)nucleotide sequences that are within a 10% variance to the respectiveSEQ ID Nos. or disclosed nucleotide sequences. The above discussionwould analogously apply to RNA sequences disclosed in this application.

Since nucleotide codons are redundant, also within the scope of thisinvention are equivalent nucleotide sequences which include: nucleotidesequences which code for the same proteins or equivalent proteins. Thus,nucleotide sequences which encode substantially the same or functionallyequivalent amino acid sequence may be used in the practice of theinvention.

The terms "BDV genomic nucleotide sequence", "p18", "recp18", "pol","recpol", "p57", "recp57", as used in relation to nucleotide sequencesare defined above, together with: (1) nucleotide sequences that arewithin an 10% variance to the respective nucleotide sequences in Table1; (2) nucleotide sequences that are capable of hybridizing to thecoding sequences of the respective nucleotide sequences, under stringenthybridization conditions, (3) nucleotide sequences coding for gp18,recp18, p57, recp57, pol, and recpol proteins, and amino acid sequencesof SEQ ID Nos. 6, 8, and 10 respectively; and (4) fragments of SEQ IDNos. 6; 8; 10; nucleotide number 1 through 53 and nucleotide number 1880through 8910 of SEQ ID NO 19 and their fragments; or other nucleotidesequences which, for example, encode proteins having substantially thesame biological characteristics/activities of gp18, recp18, p57, recp57,pol, recpol proteins, respectively. Preferably, the determinativebiological characteristic/activity is the retention of at least oneimmunoepitope. Preferably, when used in an immunoassay for BDV, theseproteins are immunoreactive with antibodies directed to BDV but notdetectably immunoreactive with non-BDV specific antibodies found in abiological sample such as a serum sample. Alternatively, the nucleotidesequences can be nucleotide probes of at least 10 nucleotides in length.Preferably, when used in a hybridization assay for BDV, these probes donot detectably hybridize to the nucleotide sequences of non-BDVorganisms which are found in a biological sample such as a serum sample.Alternatively, the nucleotide sequences hybridize to at least 10consecutive nucleotides in the coding sequences of the above listednucleotide sequences. The nucleotide sequences include a nucleotidesequence which encodes a protein containing at least 8; more preferably,5 to 6; and most preferably, 4 amino acids. Preferably, the protein isspecific to BDV or retain one or more biological functions of BDV.Examples of such biological functions are: BDV's ability to bind aparticular cellular receptor, BDV's ability to target its host cells(e.g. cells and tissues of the nervous system, bone marrow, peripheralblood, mononuclear cells or brain), and BDV's effects on the functionsof cells infected by it. The discussion herein similarly applies to p23,recp23, p80, recp80 nucleotide sequences, and the cDNA nucleotidesequence of FIG. 19, e.g. in reference to their respective SEQ ID NOsand FIG. 19.

The terms "gp18", "recp18", "p57", "recp57", "pol", and "recpol", asused in relation to proteins are, respectively, as defined abovetogether with: (1) protein variants having 95% identity, i.e.,containing amino acid sequences that have at least 95% of their aminoacids matching the sequences of SEQ ID Nos. 6, 8, and 10, respectively;(2) the functional equivalents of these proteins and their variants,respectively; and (3) the derivatives, including fragments, of gp18,recp18, p57, recp57, pol, recpol, proteins and their variants,respectively. Preferably, when used in an immunoassay for BDV, theseproteins are immunoreactive with antibodies directed to BDV but notdetectably immunoreactive with non-BDV specific antibodies found in abiological sample such as a serum sample. Alternatively, these proteinseach contains at least 8; more preferably, 5 to 6; and most preferably,4 amino acids. Preferably, the latter proteins are specific to BDV orretain one or more biological functions of BDV. Examples of suchbiological functions are: BDV's ability to bind a particular cellularreceptor, BDV's ability to target its host cells (e.g. cells and tissuesof the nervous system, bone marrow, peripheral blood, mononuclear cellsor brain), and BDV's effects on the functions of cells infected by it.The discussion herein similarly applies to p23, recp23, p80, and recp80proteins, e.g. in reference to their respective SEQ ID NOs.

Within the definition of "BDV" are BDV isotypes, strains, and BDVrelated viruses. The term "BDV proteins and their derivatives", includesBDV proteins, fragments of BDV proteins, proteins containingimmunoepitopes of BDV, variants and functional equivalents of theforegoing. gp18 and p57 are examples of BDV proteins. Preferably, theimmunoepitope is specific to BDV.

The variants can result from, e.g. substitution, insertion, or deletionof the amino acid sequences shown in Table 1. The derivatives of theproteins and their variants, include fragments of these proteins andtheir immunogenic epitopes. Preferably, each of the fragments containsat least one immunogenic epitope of BDV. More preferably, the fragmentis capable of being bound by polyclonal antibodies directed to BDV. Inthe case of antibodies which recognize linear epitopes, they generallybind to epitopes defined by about 3 to 10 amino acids. Preferably, too,each variant retains at least one immunoepitope of BDV. Preferably theimmunoepitope is specific to BDV.

Two amino acid sequences are functionally equivalent if they havesubstantially the same biological activities. The proteins may be fusedto other proteins, for example, signal sequence fusions may be employedin order to more expeditiously direct the secretion of the BDV protein.The heterologous signal replaces the native BDV signal, and when theresulting fusion is recognized, i.e. processed and cleaved by the hostcell, the BDV protein is secreted. Signals are selected based on theintended host cell, and may include bacterial, yeast, insect, mammalian,and viral sequences. For example, the native BDV signal or the herpes gDglycoprotein signal is suitable for use in mammalian expression systems.

Substitutional variants of the proteins disclosed herein are those inwhich at least one residue in the disclosed sequences has been removedand a different residue inserted in its place. Preferably, the aminoacid change is conservative. Such conservative substitutions generallyare made in accordance with the following Table 2.

                  TABLE 2                                                         ______________________________________                                        Original Residue Conservative Substitutions                                   ______________________________________                                        Ala              ser                                                          Arg              lys                                                          Asn              gln; his                                                     Asp              glu                                                          Cys              ser; ala                                                     Gln              asn                                                          Glu              asp                                                          Gly              pro                                                          His              asn; gln                                                     Ile              leu; val                                                     Leu              ile; val                                                     Lys              arg; gln; glu                                                Met              leu; ile                                                     Phe              met; leu; tyr                                                Ser              thr                                                          Thr              ser                                                          Trp              tyr                                                          Tyr              trp; phe                                                     Val              ile; leu                                                     ______________________________________                                    

Novel amino acid sequences, as well as isosteric analogs (amino acid orotherwise), are included within the scope of this invention.

A variant typically is made by site specific mutagenesis of the encodingnucleic acid, expression of the variant nucleic acid in recombinant cellculture and, optionally, purification from the cell culture for exampleby immunoaffinity adsorption on a column to which are bound polyclonalantibodies directed against the original protein from which the variantis derived.

Another class of variants are deletional variants. Deletions arecharacterized by the removal of one or more amino acid residues from theoriginal protein sequence. Typically, deletions are used to affect theoriginal protein's biological activities. However, deletions whichpreserve the biological activity or immune cross-reactivity of theoriginal protein are preferred.

Deletions of cysteine or other labile residues also may be desirable,for example in increasing the oxidative stability of the originalprotein. Deletion or substitutions of potential proteolysis sites, e.g.Arg Arg, is accomplished by deleting one of the basic residues orsubstituting one by glutaminyl or histidyl residues.

It will be understood that some variants may exhibit reduced or nobiological activity. These variants nonetheless are useful as standardsin immunoassays for BDV protein so long as they retain at least oneimmunogenic epitope of BDV protein.

It is presently believed that the three-dimensional structure of theproteins of the present invention is important to their functioning asdescribed herein. Therefore, all related structural analogs which mimicthe active structure of those formed by the compositions or proteinsclaimed herein are specifically included within the scope of the presentinvention.

Modified proteins are also within the contemplation of this patentapplication. These modifications may be deliberate, e.g., modificationsobtained through site-directed mutagenesis, or may be accidental, e.g.,as those obtained through mutation of the hosts.

Further, as is the case for all proteins, the precise chemical structuredepends on a number of factors. As ionizable amino and carboxyl groupsare present in the molecule, a particular protein may be obtained as anacidic or basic salt, or in neutral form. All such preparations whichretain their activity when placed in suitable environmental conditionsare included in the definition. Additionally, the primary amino acidsequence may be augmented by derivatization using sugar moieties(glycosylation) or by other supplementary molecules such as lipids,phosphate, acetyl groups and the like, more commonly by conjugation withsaccharides. The primary amino acid structure may also aggregate to formcomplexes, most frequently dimers. Certain aspects of such augmentationare accomplished through post-translational processing systems of theproducing host; other such modifications may be introduced in vitro. Inany event, such modifications are included in the definition so long asthe activity of the protein is not destroyed. It is expected that suchmodifications may quantitatively or qualitatively affect the activity,either by enhancing or diminishing the activity of the protein invarious assays.

Individual amino acid residues in the chain may also be modified byoxidation, reduction, or other derivatization, and the protein may becleaved to obtain fragments which retain activity. Such alterationswhich do not destroy activity do not remove the protein sequence fromthe definition. The following discusses some of the modifications infurther detail by way of example.

Thus, glycosylation variants are included within the scope of BDV. Theyinclude variants completely lacking in glycosylation (unglycosylated)and variants having at least one less glycosylated site than the nativeform (deglycosylated) as well as variants in which the glycosylation hasbeen changed. Included are deglycosylated and unglycosylated amino acidsequence variants, deglycosylated and unglycosylated BDV and gp18 havingthe native, unmodified amino acid sequence of BDV and gp18, and otherglycosylation variants, e.g. of p57. For example, substitutional ordeletional mutagenesis is employed to eliminate the N- or O-linkedglycosylation sites of BDV or gp18, e.g., an asparagine residue isdeleted or substituted for by another basic residue such as lysine orhistidine. Alternatively, flanking residues making up the glycosylationsite are substituted or deleted, even though the asparagine residuesremain unchanged, in order to prevent glycosylation by eliminating theglycosylation recognition site.

Unglycosylated protein which has the amino acid sequence of the nativeprotein is preferably produced in recombinant prokaryotic cell culturebecause prokaryotes are incapable of introducing glycosylation intopolypeptides.

Glycosylation variants are produced by selecting appropriate host cellsor by in vitro methods. Yeast, for example, introduce glycosylationwhich varies significantly from that of mammalian systems. Similarly,mammalian cells having a different species (e.g. hamster, murine,insect, porcine, bovine or ovine) or tissue origin (e.g. lung, liver,lymphoid, mesenchymal or epidermal) than the source of the BDV antigenare routinely screened for the ability to introduce variantglycosylation as characterized for example by elevated levels of mannoseor variant ratios of mannose, fucose, sialic acid, and other sugarstypically found in mammalian glycoproteins. In vitro processing of theproteins of the present invention typically is accomplished by enzymatichydrolysis, e.g. endoglycosidase digestion.

Derivatization with bifunctional agents is useful for preparingintermolecular aggregates of BDV proteins and their derivatives withpolypeptides as well as for cross-linking the protein and derivatives toa water insoluble support matrix or surface for use in the assay oraffinity purification of its ligands. In addition, a study of intrachaincross-links will provide direct information on conformational structure.Commonly used cross-linking agents include sulfhydryl reagents,1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde,N-hydroxysuccinimide esters, for example esters with 4-azidosalicylicacid, homobifunctional imidoesters including disuccinimidyl esters suchas 3,3'-dithiobis (succinimidyl-propionate), and bifunctional maleimidessuch as bis-N-maleimido-1,8-octane.

Certain post-translational derivatizations are the result of the actionof recombinant host cells on the expressed polypeptide. Glutaminyl andasparaginyl residues are frequently post-translationally deamidated tothe corresponding glutamyl and aspartyl residues. Alternatively, theseresidues are deamidated under mildly acidic conditions. Either form ofthese residues falls within the scope of this invention.

Other post-translational modifications include hydroxylation of prolineand lysine, phosphorylation of hydroxyl groups of seryl or threonylresidues, methylation of the α-amino groups of lysine, arginine, andhistidine side chains {T. E. Creighton, Proteins: Structure andMolecular Properties, W. H. Freeman & Co., San Francisco, pp 79-86(1983)}, acetylation of the N-terminal amine and, in some instances,amidation of the C-terminal carboxyl.

The claimed proteins are preferably produced using recombinanttechnologies. The nucleotide, e.g., DNA or RNA, sequences which encodethe desired polypeptides are amplified by use of e.g. the polymerasechain reaction in the case of DNA (hereinalso referred to as "PCR"), andreverse transcriptase-polymerase chain reaction (RT-PCR) in the case ofRNA. Oligonucleotide sequences to be used as primers which canspecifically bind to the ends of the regions of interest aresynthesized. After the desired region of the gene has been amplified thedesired sequence is incorporated into an expression vector which istransformed into a host cell. The nucleotide sequence is then expressedby the host cell to give the desired polypeptide which is harvested fromthe host cell. Plant, bacterial, yeast, insect, viral and mammalianexpression systems may be used. Vectors which may be used in theseexpression systems may contain fragments of plant, bacterial, yeast,insect, viral, and/or mammalian origins.

Given the teachings contained herein, one skilled in the art can createthe sequences disclosed herein, either by hand or with an automatedapparatus. As examples of the current state of the art relating topolynucleotide synthesis, one is directed to Maniatis et al., MolecularCloning--A Laboratory Manual, Cold Spring Harbor Laboratory (1984), andHorvath et al., An Automated DNA Synthesizer Employing Deoxynucleoside3'-Phosphoramidites, Methods in Enzymology 154: 313-326, 1987.

Identification of Nucleotide Sequences, Cloning, and Expression of theDisclosed Protein

Alternatively, to obtain RNA encoding the proteins disclosed herein, oneneeds only to conduct hybridization screening with labelled BDVnucleotide sequence (usually, greater than about 20, and ordinarilyabout 50 bp) in order to detect clones which contain homologoussequences in the cDNA libraries derived from cells or tissues of aparticular animal, followed by analyzing the clones by restrictionenzyme analysis and nucleic acid sequencing to identify full-lengthclones. The cell lines, cells and tissues are preferably from thenervous system, bone marrow, peripheral blood, mononuclear cells orbrain of BDV infected animals. Examples of cells from the nervous systemare: neurons, oligodendrocytes and astrocytes. The primers shown inExamples 1 to 4 and/or the methods shown therein may also be used.

If full length clones are not present in the library, then appropriatefragments are recovered from the various clones and ligated atrestriction sites common to the fragments to assemble a full-lengthclone.

The techniques shown in this section are also useful for identifying andsequencing various isotypes and strains of BDV and BDV related viruses.The present invention discloses the nucleotide sequences of two strainsof BDV; different strains of BDV may exist or arise due to mutation asin the case of human immunodeficiency virus (HIV) which constantlymutates and of which different strains are constantly being discovered.Thus, within the definition of BDV are other BDV isotypes and strains orviruses related to BDV ("BDV related viruses"). For example, the nextsection of the application describes diagnostic assays for BDV orrelated pathogenesis. The related pathogenesis include: (1) diseasescaused by BDV; (2) opportunistic or attendant diseases arising from BDVinfection; and (3) diseases caused by BDV related viruses. The BDVrelated viruses would be nonsegmented, negative-stranded, neurotropic,post transcriptionally modified (spliced) viruses which share somehomology with BDV nucleotide or amino acid sequences. Patients infectedby the BDV related viruses would manifest clinical symptoms similar toBDV infected patients, or to that of neurologic or neuropsychiatricdiseases.

Thus, DNA or RNA encoding various BDV isotypes and strains, and BDVrelated viruses, can be similarly obtained by probing libraries fromcells and tissues, especially cells of the nervous system, of animalsexhibiting clinical symptoms of BDV infection, neurologic orneuropsychiatric disease; or animals that have been purposely infectedwith BDV strains, isotypes or BDV related viruses, such as shown inExample 2. Once the DNA or RNA sequence of these strains, isotypes, orrelated viruses are known, primers based on the sequence may be used.The methods shown in Examples 1 and 2, and the primers shown therein mayalso be used to obtain the genomic nucleotide sequences.

In general, prokaryotes are used for cloning of DNA sequences inconstructing the vectors useful in the invention. For example, E. coliK12 strain 294 (ATCC No. 31446) is particularly useful. Other microbialstrains which may be used include E. coli B and E. coli X1776 (ATCC No.31537). These examples are illustrative rather than limiting.Alternatively, in vitro methods of cloning, e.g. polymerase chainreaction, are suitable.

The proteins of this invention may be expressed directly in recombinantcell culture as an N-terminal methionyl analogue, or as a fusion with apolypeptide heterologous to the hybrid/portion, preferably a signalsequence or other polypeptide having a specific cleavage site at theN-terminus of the hybrid/portion. For example, in constructing aprokaryotic secretory expression vector for portion/fragment of BDVprotein, the native BDV signal is employed with hosts that recognizethat signal. When the secretory leader is "recognized" by the host, thehost signal peptidase is capable of cleaving a fusion of the leaderpolypeptide fused at its C-terminus to the desired mature BDV protein.For host prokaryotes that do not process the BDV signal, the signal issubstituted by a prokaryotic signal selected, for example, from thegroup of the alkaline phosphatase, penicillinase, lpp or heat stableenterotoxin II leaders. For yeast secretion the BDV signal may besubstituted by the yeast invertase, alpha factor or acid phosphataseleaders. In mammalian cell expression, the native signal is satisfactoryfor mammalian BDV, although other mammalian secretory protein signalsare suitable, as are viral secretory leaders, for example the herpessimplex gD signal.

The proteins of the present invention may be expressed in any host cell,but preferably are synthesized in mammalian hosts. However, host cellsfrom prokaryotes, fungi, yeast, insects and the like are also are usedfor expression. Exemplary prokaryotes are the strains suitable forcloning as well as E. coli W3110 (F-λ-A-prototrophic, ATTC No. 27325),other enterobacteriaceae such as Serratia marcescans, bacilli andvarious pseudomonads.

Expression hosts typically are transformed with DNA encoding theproteins of the present invention which has been ligated into anexpression vector. Such vectors ordinarily carry a replication origin(although this is not necessary where chromosomal integration willoccur). Expression vectors also include marker sequences which arecapable of providing phenotypic selection in transformed cells, as willbe discussed further below. For example, E. coli is typicallytransformed using pBR322, a plasmid derived from an E. coli species{Bolivar, et al., Gene 2:95 (1977)}. pBR322 contains genes forampicillin and tetracycline resistance and thus provides easy means foridentifying transformed cells, whether for purposes of cloning orexpression. Expression vectors also optimally will contain sequenceswhich are useful for the control of transcription and translation, e.g.,promoters and Shine-Dalgarno sequences (for prokaryotes) or promotersand enhancers (for mammalian cells). The promoters may be, but need notbe, inducible; even powerful constitutive promoters such as the CMVpromoter for mammalian hosts may produce BDV proteins without host celltoxicity. While it is conceivable that expression vectors need notcontain any expression control, replicative sequences or selectiongenes, their absence may hamper the identification of transformants andthe achievement of high level peptide expression.

Promoters suitable for use with prokaryotic hosts illustratively includethe β-lactamase and lactose promoter systems {Chang et al., Nature275:615 (1978); and Goeddel et al., Nature 281:544 (1979)}, alkalinephosphatase, the tryptophan (trp) promoter system (Goeddel, NucleicAcids Res. 8:4057 (1980) and EPO Appln. Publ. No. 36,776) and hybridpromoters such as the tac promoter {H. de Boer et al., Proc. Natl. Acad.Sci. USA 80:21-25 (1983)}. However, other functional bacterial promotersare suitable. Their nucleotide sequences are generally known, therebyenabling a skilled worker operably to ligate them to DNA encoding theproteins of the present invention {Siebenlist et al., Cell 20:269(1980)} using linkers or adaptors to supply any required restrictionsites. Promoters for use in bacterial systems also will contain aShine-Dalgarno (S.D.) sequence operably linked to the DNA encoding theproteins of the present invention

In addition to prokaryotes, eukaryotic microbes such as yeast orfilamentous fungi are satisfactory. Saccharomyces cerevisiae is the mostcommonly used eukaryotic microorganism, although a number of otherstrains are commonly available. The plasmid YRp7 is a satisfactoryexpression vector in yeast {Stinchcomb, et al., Nature 282:39 (1979);Kingsman et al., Gene 7:141 (1979); Tschemper et al., Gene 10:157(1980)}. This plasmid already contains the trp1 gene which provides aselection marker for a mutant strain of yeast lacking the ability togrow in tryptophan, for example ATCC no. 44076 or PEP4-1 {Jones,Genetics 85:12 (1977)}. The presence of the trp1 lesion as acharacteristic of the yeast host cell genome then provides an effectiveenvironment for detecting transformation by growth in the absence oftryptophan. Alternatively, viral expression vectors such as retroviralvectors, baculoviral vectors and Semliki Forest viral vectors are used.The expression hosts of these vectors are known in the art.

Suitable promoting sequences for use with yeast hosts include thepromoters for 3-phosphoglycerate kinase {Hitzeman et al., J. Biol. Chem.255:2073 (1980)} or other glycolytic enzymes {Hess et al., J. Adv.Enzyme Reg. 7:149 (1968); and Holland, Biochemistry 17:4900 (1978)},such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase,pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphateisomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphateisomerase, phosphoglucos isomerase, and glucokinase.

Other yeast promoters, which are inducible promoters having theadditional advantage of transcription controlled by growth conditions,are the promoter regions for alcohol dehydrogenase 2, isocytochrome C,acid phosphatase, degradative enzymes associated with nitrogenmetabolism, metallothionein, glyceraldehyde-3-phosphate dehydragenase,and enzymes responsible for maltose and galactose utilization. Suitablevectors and promoters for use in yeast expression are further describedin R. Hitzeman et al., European Patent Publication No. 73,657A.

Expression control sequences are known for eukaryotes. Virtually alleukaryotic genes have an AT-rich region located approximately 25 to 30bases upstream from the site where transcription is initiated. Anothersequence found 70 to 80 bases upstream from the start of transcriptionof many genes is a CXCAAT region where X may be any nucleotide. At the3' end of most eukaryotic genes is an AATAAA sequence which may be thesignal for addition of the poly A tail to the 3' end of the codingsequence. All of these sequences may be inserted into mammalianexpression vectors.

Suitable promoters for controlling transcription from vectors inmammalian host cells are readily obtained from various sources, forexample, the genomes of viruses such as polyoma virus, SV40, adenovirus,MMV (steroid inducible), retroviruses (e.g. the LTR of BDV), hepatitis-Bvirus and most preferably cytomegalovirus, or from heterologousmammalian promoters, e.g. the beta actin promoter. The early and latepromoters of SV40 are conveniently obtained as an SV40 restrictionfragment which also contains the SV40 viral origin of replication.{Fiers et al., Nature 273:113 (1978)}. The immediate early promoter ofthe human cytomegalovirus is conventionally obtained as a HindIII Erestriction fragment. {Greenaway, P. J. et al., Gene 18:355-360 (1982)}.

Transcription of a DNA encoding the proteins of the present invention byhigher eukaryotes is increased by inserting an enhancer sequence intothe vector. Enhancers are cis-acting elements of DNA, usually about from10-300 bp, that act on a promoter to increase its transcription.Enhancers are relatively orientation and position independent havingbeen found 5' {Laimins et al., Proc. Natl. Acad. Sci., 78:993 (1981)}and 3' {Lusky, M. L., et al., Mol. Cell Bio. 3:1108 (1983)} to thetranscription unit, within an intron {Banerji, J. L. et al., Cell 33:729(1983)} as well as within the coding sequence itself {Osborne, T. F., etal., Mol. Cell Bio. 4:1293 (1984)}. Many enhancer sequences are nowknown from mammalian genes (globin, elastase, albumin, α-fetoprotein andinsulin). Typically, however, one will use an enhancer from a eukaryoticcell virus. Examples include the SV40 enhancer on the late side of thereplication origin (bp 100-270), the cytomegalovirus early promoterenhancer, the polyoma enhancer on the late side of the replicationorigin, and adenovirus enhancers.

Expression vectors used in eukaryotic host cells (yeast, fungi, insect,plant, animal, human or nucleated cells from other multicellularorganisms) will also contain sequences necessary for the termination oftranscription which may affect mRNA expression. These regions aretranscribed as polyadenylated segments in the untranslated portion ofthe mRNA. The 3' untranslated regions also include transcriptiontermination sites.

Expression vectors may contain a selection gene, also termed aselectable marker. Examples of suitable selectable markers for mammaliancells are dihydrofolate reductase (DHFR), thymidine kinase (TK) orneomycin. When such selectable markers are successfully transferred intoa mammalian host cell, the transformed mammalian host cell is able tosurvive if placed under selective pressure. There are two widely useddistinct categories of selective regimes. The first category is based ona cell's metabolism and the use of a mutant cell line which lacks theability to grow independent of a supplemented media. Two examples areCHO DHFR-cells and mouse LTK cells. These cells lack the ability to growwithout the addition of such nutrients as thymidine or hypoxanthine.Because these cells lack certain genes necessary for a completenucleotide synthesis pathway, they cannot survive unless the missingnucleotides are provided in a supplemented media. An alternative tosupplementing the media is to introduce an intact DHFR or TK gene intocalls lacking the respective genes, thus altering their growthrequirements. Individual cells which were not transformed with the DHFRor TK gene will not be capable of survival in non-supplemented media.

The second category of selective regimes is dominant selection whichrefers to a selection scheme used in any cell type and does not requirethe use of a mutant cell line. These schemes typically use a drug toarrest growth of a host cell. Those cells which are successfullytransformed with a heterologous gene express a protein conferring drugresistance and thus survive the selection regimen. Examples of suchdominant selection use the drugs neomycin {Southern et al., J. Molec.Appl. Genet. 1:327 (1982)}, mycophenolic acid {Mulligan et al., Science209:1422 (1980)} or hygromycin {Sugden et al., Mol. Cell. Biol.5:410-413 (1985)}. The three examples given above employ bacterial genesunder eukaryotic control to convey resistance to the appropriate drugG418 or neomycin (geneticin), xgpt (mycophenolic acid) or hygromycin,respectively.

"Amplification" refers to the increase or replication of an isolatedregion within a cell's chromosomal DNA. Amplification is achieved usinga selection agent, e.g. methotrexate (MTX) which inactivates DHFR.Amplification or the making of successive copies of the DHFR generesults in greater amounts of DHFR being produced in the face of greateramounts of MTX. Amplification pressure is applied notwithstanding thepresence of endogenous DHFR, by adding ever greater amounts of MTX tothe media. Amplification of a desired gene can be achieved bycotransfecting a mammalian host cell with a plasmid having a DNAencoding a desired protein and the DHFR or amplification gene permittingcointegration. One ensures that the cell requires more DHFR, whichrequirement is met by replication of the selection gene, by selectingonly for cells that can grow in the presence of ever-greater MTXconcentration. So long as the gene encoding a desired heterologousprotein has cointegrated with the selection gene replication of thisgene gives rise to replication of the gene encoding the desired protein.The result is that increased copies of the gene, i.e. an amplified gene,encoding the desired heterologous protein express more of the desiredprotein.

Suitable eukaryotic host cells for expressing the proteins includemonkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); humanembryonic kidney line (293 or 293 cells subcloned for growth insuspension culture, {Graham, F. L. et al., J. Gen Virol. 36:59 (1977)};baby hamster kidney cells (BHK, ATCC CCL 10); chinese hamsterovary-cells-DHFR {CHO, Urlaub and Chasin, Proc. Natl. Acad. Sci., (USA)77:4216, (1980)}; mouse sertoli cells {TM4, Mather, J. P., Biol. Reprod.23:243-251 (1980)}; monkey kidney cells (CV1 ATCC CCL 70); african greenmonkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinomacells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34);buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138,ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor(MMT 060562, ATCC CCL51); TRI cells {Mather, J. P., et al., Annals N.Y.Acad. Sci. 383:44-68 (1982)}; and C₆ glial cell (ATCC CCL 107).

Construction of suitable vectors containing the desired coding andcontrol sequences employ standard ligation techniques. Isolated plasmidsor DNA fragments are cleaved, tailored, and religated in the formdesired to form the plasmids required.

For analysis to confirm correct sequences in plasmids constructed, theligation mixtures are used to transform E. coli K12 strain 294 (ATCC31446) and successful transformants selected by ampicillin ortetracycline resistance where appropriate. Plasmids from thetransformants are prepared, analyzed by restriction and/or sequenced bythe method of Messing et al., Nucleic Acids Res. 9:309 (1981) or by themethod of Sanger et al., Proc. Natl. Acad. Sci., (USA), 74:5463 (1977).

Host cells are transformed with the expression vectors of this inventionand cultured in conventional nutrient media modified as appropriate forinducing promoters, selecting transformants or amplifying the genesencoding the desired sequences. The culture conditions, such astemperature, pH and the like, are those previously used with the hostcell selected for expression, and will be apparent to the ordinarilyskilled artisan.

The host cells referred to in this disclosure encompass cells in vitroculture as well as cells which are within a host animal.

Diagnostic, Prognostic, and Monitoring Uses of BDV Proteins and TheirDerivatives

Another aspect of the present invention presents assays for detectingligands, e.g., in the biological samples of a test organism, which bindBDV protein(s) or derivatives thereof. These assays are useful asdiagnostic tests for: (1) infection by BDV or related pathogenesis; and(2) neurologic and neuropsychiatric disease not due to BDV infection.

The preferred assays are immunoassays which detect antibodies to BDVproteins or its derivatives that are antigenic (herein referred to as"BDV antigen"). The test organism can be human or other animals, such ascats, fowls, ostriches, and horses. The biological samples may bebiological fluids such as whole blood, serum, plasma, cerebral spinalfluid, or synovial fluid. Preferably, BDV proteins or its derivativesare used to detect the ligand by binding to it. Preferably, the ligandis an antibody directed to the polypeptides, and BDV antigens are usedto detect the antibody. For example, the assay can be used to detectantibodies against BDV in biological fluids.

Alternatively, antibodies to BDV protein(s) or their derivatives can beused to screen for BDV proteins, e.g., in the biological samples of atest organism. Similarly, the alternative detection of antibodies orantigen applies to each of the assay formats described below.

Thus, an example of the assay is an enzyme immunoassay. In an example ofa direct assay, these polypeptides serve as antigens and are attached toa solid phase and then incubated with patient sera. Human serum orplasma is preferably diluted in a sample diluent before incubation. Ifantibodies to BDV are present in the sample they will form anantigen-antibody complex with the polypeptides and become affixed to thesolid phase.

After the antigen-antibody complex has formed, unbound materials andreagents are removed by washing the solid phase and the antigen-antibodycomplex is reacted with a solution containing labelled antibodiesdirected against the first type of antibody. For example, the labelledantibody can be horseradish peroxidase-labeled goat antibody. Thisperoxidase labelled antibody then binds to the antigen-antibody complexalready affixed to the solid phase. In a final reaction the horseradishperoxidase is contacted with o-phenylenediamine and hydrogen peroxidewhich results in a yellow-orange color. The intensity of the color isproportional to the amount of antibody which initially binds to thepolypeptide affixed to the solid phase.

Another assay format provides for an antibody-capture assay in whichanti-immunoglobulin antibody on the solid phase captures the patient'santibody, which is then reacted with the BDV antigen. The application ofthis format is similar to the serological assay of Lyme disease taughtin Berardi et al., J. Infect. Dis. 158:754-760 (1988). If antibody toBDV is present, it captures the BDV antigen, and the bound BDV antigenis detected by means of labelled polyclonal or monoclonal antibodiesdirected against the BDV antigen. The antibody-capture assay isparticularly useful for and can increase the sensitivity of detection ofIgM and IgG to BDV antigens. In an example of this assay, the fluidsample is first contacted with a solid support containing a boundantibody capable of binding the mu-chain of IgM or the gamma-chain ofIgG antibodies. Specific antibody is detected by reacting this with theBDV antigens followed by non-human antibody to the BDV antigens. Thenon-human antibody is generally labelled for detection. It is believedthat this antibody-capture immunoassay format will have increasedsensitivity, especially for IgM. Alternatively, one can forego thenon-human antibody and instead label the BDV antigens for directdetection.

Another assay format provides for an immunodot assay for identifying thepresence of an antibody that is immunologically reactive with specificBDV antigens by contacting a sample with the BDV antigens bound to asolid support under conditions suitable for complexing the antibody withthe BDV antigens and detecting the antibody-antigen complex by reactingthe complex.

Suitable methods and reagents for detecting an antibody-antigen complexin an assay of the present invention are commercially available or knownin the relevant art. For example, the detector antibodies orpolypeptides may be labelled with enzymatic, radioisotopic, fluorescent,luminescent, or chemiluminescent label. These labels may be used inhapten-labelled antihapten detection systems according to knownprocedures, for example, a biotin-labelled antibiotin system may be usedto detect an antibody-antigen complex.

In all of the assays, the sample is preferably diluted before contactingthe BDV antigen absorbed on a solid support. Solid support materials mayinclude cellulose materials, such as paper and nitrocellulose; naturaland synthetic polymeric materials, such as polyacrylamide, polystyrene,and cotton; porous gels such as silica gel, agarose, dextran andgelatin; and inorganic materials such as deactivated alumina, magnesiumsulfate and glass. Suitable solid support materials may be used inassays in a variety of well known physical configurations, includingmicrotiter wells, test tubes, beads, strips, membranes, andmicroparticles. A preferred solid support for a non-immunodot assay is apolystyrene microwell, polystyrene beads, or polystyrene microparticles.A preferred solid support for an immunodot assay is nitrocellulose ornylon membrane.

In particular, the invention presents an ELISA which is a rapid,sensitive, and inexpensive diagnostic test. The preferred ELISAs arebased on recombinant BDV proteins recp40, recp23, and recp18. Theseassays are more sensitive and rapid than prior art methods employed forserologic diagnosis of infection, such as Western blot, indirectimmunofluorescent test or immunoprecipitation.

Examples of the neurologic and neuropsychiatric diseases that can bediagnosed include diseases of the nervous system such as schizophrenia,depressive disorders (e.g., bipolar depression), multiple sclerosis andAIDS-related encephalopathy. The finding is based on applicants'analysis of the art. Although the virus has not been recovered fromhuman subjects, antibodies reactive with BDV proteins have been found inpatients with bipolar depression, schizophrenia, or AIDS-relatedencephalopathy {Bode, L., et al., Arch. Virol. Suppl., 7:159-167 (1993);Bode, L., et al., Lancet, ii:689 (1988) and Rott, R., et al., Science228:755-756 (1985)}. BDV has a unique tropism for specific brainregions. Viral nucleic acids and disease-specific proteins have beenfound in highest concentrations in the hippocampus and limbic circuits,prefrontal and cingulate cortices, and brainstem nuclei {Carbone, K., etal., J. Neuropathol. Exp. Neurol., 50:205-214 (1991); Ludwig, H., etal., Prog. Med. Virol. 35:107-151 (1988) and Solbrig, M. V., et al.,abstr. 10, Abstr. 1992 Am. Acad. Neurol. Annu. Meet., (1992)}. Three BDVproteins, p40, p23 and gp18 (disclosed in Example 2 below) have beenidentified in infected cells and tissues {Ludwig, H., et al., Prog. Med.Virol. 35:107-151 (1988) and Thiedemann, N., et al., J. Gen. Virol.,73:1057-1064 (1992)}. cDNAs for p40 {Lipkin, W. I., et al., Proc. Natl.Acad. Sci. USA, 87:4184-4188 (1990); McClure, M. A., et al., J. Virol.,66:6572-6577 (1992) and Pyper, J. M., et al., Virology, 195:229-238(1993)} and p23 {Lipkin, W. I., et al., Proc. Natl. Acad. Sci. USA,87:4184-4188 (1990); Thierer, J., et al., J. Gen. Virol., 73:413-416(1992) and VandeWoude, S., et al., Science, 250:1276-1281 (1990)} havebeen isolated, and complementary sequences to open reading frames (ORFs)for these proteins have been mapped to the viral genome {Briese, T., etal., Proc. Natl. Acad. Sci. USA 91:4362-4366 (1994) which isincorporated into Example 1 of this application; and Cubitt, B., et al.,J. Virol., 68:1382-1996 (1994)}.

The assay can also be used to monitor the diseases by monitoring thetiter of such ligands. The titer of the ligands, and the specific viralproteins that it is immunoreactive with, can also be prognosticative ofthe diseases.

Thus, an application of this invention may involve contacting the testsubject's biological sample, such as serum, with a panel consisting ofdifferent immunogenic fragments of BDV protein or its derivatives. Theseproteins may be synthetic or native proteins, though recombinantproteins are preferred. Such a panel may consist of, for example,recp23, recp40, recp57, recpol and recp18. If the serum isimmunoreactive with at least one of the fragments, it indicates that thetest subject may either be suffering from (1) BDV or relatedpathogenesis; or (2) neurologic and neuropsychiatric disease not due toBDV infection. Further, given a fixed amount of sample tested, theamount (i.e. percentage) of ligands immunoreactive with the BDV proteinsmay also be indicative of the severity of the disease and thus itsprognosis. Generally, the higher the percentage of ligands that areimmunoreactive, the more severe the disease and the poorer theprognosis. Thus, the assay may also be used to monitor the progress ofthe disease. In particular, if the test subject is undergoing treatmentfor the disease, the assay may be used to monitor the efficacy of thedrug and treatment regimen. Such monitoring may involve assaying for theligand titer and/or the specific BDV immunogenic epitopes which theligand binds to.

Hybridization Diagnostic Assays

Oligonucleotides ("probes") that are unique, or relatively unique to BDVin a test sample, are useful for diagnosing BDV infections. Nucleotidehybridization assay may be used, whereby nucleic acids from a patient'sbiological sample are contacted to the primers or BDV restrictionfragments under hybridization condition, and the hybridization productsare detected. This method could be used to detect viral genomic RNA ormRNA. Conventional Western or Northern Blot analysis, RT-PCR or PCR andligase chain reaction (LCR) may be used as the basis of the assay, thesetechniques are known to those skilled in the art. PCR and LCR techniquesare widely available in the art. For example, the basic PCR techniquesare described in U.S. Pat. Nos. 4,683,202; 4,683,195; 4,800,159; and4,965,188. The basic LCR techniques are described in EPA-320,308;EPA-439,182; EPA-336,731; WO 89/09835; WO 89/12696, and WO 90/01069.

Since the present invention presents the full nucleotide sequence of thegenomic BDV nucleotide sequence, these probes can be identified bycomparing this sequence with the sequences of other organisms which maycontaminate a test sample. Such comparison can be conducted as describedin Example 1 below or using methods known in the art. The probespreferably contain at least 10 contiguous nucleotides or at least 30contiguous nucleotides with at least 60% homology along the length ofthe BDV nucleotide sequence being compared. Examples of such probes andmethods for conducting the PCR for detection are as described inExamples 1 and 2.

Assay Kits

The present invention also encompasses immunoassay kits containing BDVantigen(s), preferably each antigen per container, in a concentrationsuitable for use in immunoassay. In the kits, the BDV antigens may bebound to a solid support and where needed, the kits may include samplepreparation reagents, wash reagents, detection reagents and signalproducing reagents.

Also included are assay kits for nucleotide hybridization assays whichinclude probes which are specific for BDV or its derivatives. The kitsmay also include sample preparation reagents, wash reagents, detectionreagents and signal producing reagents.

Therapeutic Uses of Antibodies Directed to BDV Proteins and TheirDerivatives

Another aspect of the invention presents methods, using antibodiesdirected to BDV proteins or derivatives, for treating: (1) BDV infectionor related pathogenesis; and (2) neurologic and neuropsychiatric diseasenot due to BDV infection. Examples of such antibodies are those specificto gp18 and p57. The antibodies may be administered using methods knownin the art. Preferably, this involves passive administration of theseantibodies, such as those described in Example 4.

Peptides Useful for Diagnostics and Therapeutics

Another aspect of the invention presents peptides e.g. the truncatedfragments and peptides disclosed in "EXAMPLE 5", below, containing atleast one BDV immunoepitope. These peptides can be used in diagnosticassays to detect the presence of a patient's antibodies agaisnt BDV.Thus, the peptides are useful for the assays described in the section:"Diagnostic, Prognostic, and Monitoring Uses of BDV proteins and theirderivatives". For example, as shown in Example 3 below, recp40, recp23,and recp18 have proved useful for detecting BDV infections. Thus, theepitopes of these recombinant proteins can be mapped, and smallerpeptides containing these epitopes and routinely tested for theirimmunoreactivity with antibodies to BDV, e.g. using the ELISA methodshown in Example 3.

The above peptides can also be used to raise antibodies that may serveas therapeutics against BDV infections such as shown in Example 4 and asdescribed in the section: "Therapeutic Uses of Antibodies Directed toBDV proteins and Their Derivatives". Examples of methods forsynthesizing peptide fragments are described in Stuart and Young in"Solid Phase Peptide Synthesis", 2nd ed., Pierce Chemical Co. (1984). Itis contemplated that antibodies which precipitate BDV viral particleswould be useful for therapeutic uses. In particular, these antibodiesare raised against proteins, and their fragments, expressed on thesurface of BDV. It is further contemplated that antibodies against gp18,p57 and their fragments, especially antibodies that precipitate BDVviral proteins would be useful for treating or preventing the disease(1) BDV infection or related pathogenesis; and (2) neurologic andneuropsychiatric disease not due to BDV infection.

Thus, fragments of BDV proteins, in particular gp18 and p57 and theirfragments, can be made starting from either end of their C-termini andNH₂ -termini. For example, these fragments can be tested according tothe ELISA method shown in Example 3 against, e.g. sera from horses,rats, or human patients infected with BDV. The fragments that react withthe sera would be useful for detecting the disease and would be usefulfor raising therapeutic antibodies to treat the disease. Since differentanimals may react to different epitopes of BDV proteins, one may eventailor the screening test by using the serum from the same species ofanimal for which one seeks to develop an assay or therapeutic. Forexample, if one is seeking a diagnostic test or therapeutic for humans,the sera tested will be preferably that from human patients. Included inthis invention are other methods, known in the art, for identifying theimmunoreactive epitopes of a protein and raising antibodies thereto.Further, since antibodies which are immunoreactive with BDV protein mayalso be found in the sera of patients with neurologic andneuropsychiatric disease not necessarily due to BDV infection, the abovepeptides and antibodies raised thereto may also find usefulness indiagnosing, monitoring and treating these patients. Additionally, thesepeptides may be identified by their immunoreactivity with sera frompatients suffering from neurologic and neuropsychiatric disease not dueto BDV infection. Thus, as described in this application, the disease,patient sera to be tested, the diagnostic, monitoring and therapeuticuses are not limited to BDV, and include (1) BDV infection or relatedpathogenesis; and (2) neurologic and neuropsychiatric disease not due toBDV infection. Further, one can screen for therapeutic ligands orchemicals which bind these peptides. These therapeutic chemicals thenmay be tested for their therapeutic effect against the above diseases.Other ligands or chemicals which bind the therapeutic ligands orchemicals can be tested for their ability to bind patients' antisera orantibodies and are thus useful as diagnostics for the diseases.

Preferably, the above peptides and antibodies are also respectivelytested for their crossreactivity with antibodies raised by and proteinsfrom organisms unrelated to the above diseases but commonly found in thetest sample (e.g. patient's biological sample). Peptides and antibodiesthat are highly non-specific are preferably not used. To obtain peptidesof high specificity, one may also compare the amino acid sequence of BDVprotein with that of known contaminating proteins in the test sample.The fragments that are unique, or relatively so, to BDV are then chosenfor further screening as described above, e.g. for immunoreactivity withpatient's test sample. These comparison can also be done on thenucleotide sequence level.

Method for Producing Antibodies to BDV and its Derivatives

Besides whole immunoglobulins, antibodies herein include antigen bindingfragments of the immunoglobulins. Examples of these fragments are Fab,F(ab')2 and Fv. Such fragments can be produced by known methods. Unlessotherwise indicated, antibodies herein also include: polyclonal andmonoclonal antibodies, monospecific antibodies, and antisera whichincludes monospecific antisera.

Antibodies to BDV proteins and their derivatives can be produced usingstandard procedures known in the art. For example, antibodies can beproduced by inoculating a host animal such as a rabbit, rat, goat,mouse, etc., with BDV proteins and their derivatives. Beforeinoculation, the polypeptides or fragments may be first conjugated withkeyhole limpet hemocyanin (KLH) or bovine serum albumin (BSA). After anappropriate time period for the animal to produce antibodies to thepolypeptides or fragments, the anti-serum of the animal is collected andthe polyclonal antibodies separated from the anti-serum using techniquesknown in the art. Monoclonal antibodies can be produced by the methoddescribed in Kohler and Milstein (Nature, 256:495-497, 1975) byimmortalizing spleen cells from an animal inoculated with thepolypeptides or fragments thereof. The immortalization of the spleencell is usually conducted by fusing the cell with an immortal cell line,for example, a myeloma cell line, of the same or different species asthe inoculated animal. The immortalized fused cell can then be clonedand the cell screened for production of the desired antibody.

The antibodies may also be recombinant monoclonal antibodies producedaccording to the methods disclosed in Reading, U.S. Pat. No. 4,474,893,or Cabilly et al., U.S. Pat. No. 4,816,567. The antibodies may also bechemically constructed according to the method disclosed in Segel etal., U.S. Pat. No. 4,676,980.

While the invention is demonstrated using mouse monoclonal antibodiesand rat monospecific antisera, the invention is not so limited. In fact,human antibodies may be used and may prove to be preferable. The latteris especially so if the antibodies are used as therapeutics for humans,as there would be less immunorejection from the human patients receivingthese antibodies. Such antibodies can be obtained by using humanhybridomas {Cote et al., Monoclonal Antibodies and Cancer Therapy, AlanR. Liss, p. 77 (1985)}. In fact, according to the invention, techniquesdeveloped for the production of chimeric antibodies {Morrison et al.,Proc. Natl. Acad. Sci., 81:6851 (1984); Neuberger et al., Nature, 312:604 (1984); Takeda et al., Nature, 314: 452 (1985)} by splicing thegenes from a mouse antibody molecule of appropriate antigen specificitytogether with genes from a human antibody molecule of appropriatebiological activity (such as ability to activate human complement andmediate antibody-dependent cell-mediated cytotoxicity) can be used; suchantibodies are within the scope of this invention.

Vaccine

By providing the nucleotide and amino acid sequences of the BDV genomeand BDV proteins, respectively, this application enables the productionof recombinant BDV {e.g. using the technique shown in Schnell, M. J.,EMBO J., 13: 4195-4203 (1994)} which can then be attenuated, e.g. bymutagenesis, heat or formaldehyde treatment, to be used as vaccineagainst (1) BDV infection or related pathogenesis; and (2) neurologicand neuropsychiatric disease not due to BDV infection. BDV sequences,their mutagenized sequences or fragments thereof, may be administered,e.g. by direct injection, or incorporated into a vector and administerede.g. by direct injection, into patients. Examples of the fragments arethe truncated fragments and peptides disclosed in "EXAMPLE 5", below.The injections may be by means of a gene gun. gp18, p57, pol, andproteins produced by the mutagenized or fragmented sequences may alsoserve as vaccines. Proteinaceous vaccines may be delivered orally,intravenously, intraperitoneally, or intramuscularly. The vaccine mayalso be contained in a physiologically compatible solution.

BDV Viral Vector Based Delivery System

Another aspect of the invention presents: (A) a BDV-mediated genetransfer for the incorporation and expression of eukaryotic orprokaryotic foreign genes into another eukaryotic or prokaryotic host;and (B) an in vitro BDV-mediated delivery of gene(s) or chemical(s) to atarget cell.

In Method A, one or more desired genes are inserted into the BDV viralvector. The desired gene transfer can be achieved through in vitrotransfection of a cell or cell line by the resulting BDV viral vector.The transfected cell or cell line thus expresses the gene(s) of interestand the expression product(s) are harvested. Alternatively, thetransfected cell or cell line is later transplanted into a host, e.g. ananimal such as a human, in need of the gene product(s). In this case,the gene(s) is expressed in vivo. The generation of infectiousnon-segmented, neurotropic, negative-stranded RNA virus entirely fromcloned cDNA, has been described in the case of rabies virus {Schnell, M.J., et al., EMBO J., 13(18): 4195-4203 (1994)}. The insertion of foreigngene(s) into the BDV viral vector is based on prior art teachings forother viral vectors, which may include insertion of promoters orregulators to control expression of the foreign gene(s). Thetransfection and gene therapy is similarly based on prior art teachingfor viral vectors. Such teachings abound, see e.g., U.S. Pat. No.5,219,740 to Miller et al., Jun. 15, 1993; U.S. Pat. No. 5,256,553 toOverell, Oct. 26, 1993; and WO 91/12329, assigned to Board of Regents,the University of Texas System, international publication date, Aug. 22,1991.

Method B utilizes the unique tropism of BDV for specific regions andcells of the nervous systems, e.g. neural cells. Thus, BDV vector can beused for in vivo delivery of chemicals or desired genes to theseregions. For example, infectious recombinant BDV containing the gene ofinterest can be used to infect the specific target cells of BDV in ahost animal. The host can be a human suffering from deficiency, lack of,or a malfunctioning of the gene product. The general gene therapymethods can be based on prior art teaching e.g. the references cited forMethod A, such as WO 91/12329. In the case of BDV viral vectors, thesegenes can be those responsible for the survival, proliferation, andproper functioning of the nervous system. For example, inneurodegenerative diseases, the cells in the patients' nervous systemsuffer premature death, and these cells are not regenerated, eventuallycausing the patients to die. The inserted gene(s) may supplement orreplace the dysfuntional gene(s) in these patients to provide geneproduct(s) necessary for continued survival and proliferation of thesecells. Examples of the inserted genes include genes coding for:neurotransmitters, cytokines, growth factors, receptors for theforegoing, enzymes for activation of therapeutic drugs administered tothe patients.

Alternatively, the viral vector may contain a nucleotide sequence codingfor a toxin. These vectors would infect the host's cells in vivo,express the toxin and kill the infected cells. The targeted cells arepreferably neoplastic cells, or cells infected by or harboringpathogenic organisms. The vector is preferably further designed toselectively target these cells over normal cells. One means to targetthe desired cells is by localized injection of the recombinant virus,containing the desired gene, near the target of interest. However, forBDV based gene therapy, the vector or recombinant virus may be deliveredperipherally, i.e. into subcutaneous tissue, peripheral nerve, orintramuscularly. The neurotropism of the recombinant virus allows it tomigrate towards cells of the nervous system to transfect or infect them.

The BDV viral vector is an especially good vehicle for gene therapy andin vivo chemical delivery. It has several advantages over the viralvectors known in the art, the most common of which are retroviralvectors. Retroviral vectors require replication of its host cells fortransfection. Therefore, retroviral vectors can only be used withdividing/mitotic cells. In contrast, BDV vectors are autonomous,self-replicating vectors and thus can transfect both dividing andnon-dividing cells. Thus, BDV is particularly effective for transfectingnerve cells that normally do not divide and for which BDV is tropic.

Further, BDV does not have a latent stage in its lifecycle, aftertransfecting a host cell. It thus will continue to express the desiredgene once it has transfected a cell. This is unlike some viral vectorscurrently used in the art, such as the herpes viral vector that mayenter a latent stage after transfection and thus not express the desiredgene product in the transfected cell. BDV is also unique in that it is aslow growing virus and is not lytic. Thus, chances of the virus lysingand killing the host cells are nonexistent.

As a further safeguard, the BDV viral vectors may be made infective butreplication-defective, rendering them useful vectors which are unable toproduce infective virus, following introduction into a cell. Forinitiation of productive infection of BDV, a nucleocapsid containing BDVgenomic RNA is required, from which primary transcription of mRNAs andensuing autonomous and regulated expression of all BDV proteins occurs.Thus, to render the viral vector replication-defective, one may mutatethe nucleocapsid protein produced by recombinant virus to preventencapsidation of newly synthesized genomic RNA. Additionally, the hostcell should preferably be devoid of infectious helper virus which mayassist in replication of the BDV.

Further, unlike retroviruses and herpes viruses, BDV does not causedisease in and of itself. The deleterious effect of BDV infection isactually caused by the host's immune-mediated rejection of BDV and BDVantigen expressed on infected cells. The rejection involves cellularimmune response which activates the host's effector lymphocytes whichthen kill the transfected cells. Antibodies appear not to be asimportant in the host's immune response. Thus, one means to avoid Bornadisease is to interfere with, avoid, or suppress the host's ability torecognize or mount an immune response to BDV infected cells. Forexample, immune response in the host is triggered when T lymphocytesrecognize a complex of major histocompatibility complex (MHC) andforeign antigen (in this case, BDV proteins) expressed on the hostcell's surface. Thus, to reduce the host's immune response, one maychoose to interfere with or prevent the expression of MHC on thetransfected cells. This may be achieved by inserting, into the BDV viralvector, a nucleotide sequence which codes for a mRNA (i.e. an antisensemRNA) which would bind the mRNA coding for the component of MHC("mRNA_(MHC) ") and prevent the translation and expression of MHC in thetransfected cell. Absent MHC, the BDV antigens will not be presented onthe host cell surface to trigger immune-mediated rejection in the host.Alternatively, other methods known in the art may be used to avoid theimmune rejection of BDV transfected cells.

EXAMPLE 1 Cloning and Sequencing of Genomic RNA from Borna Disease Virus(BDV) Particles

The studies in this example and Example 2, except with regard to p57,are also described in Briese, T., et al., Proc. Natl. Acad. Sci., USA,91:4362-4366 (1994) and Kliche, S., et al., J. Virol., 68: 6918-6923(1994), respectively, both of which are hereby incorporated by referencein their entirety. In this example, the BDV genome was cloned to revealantisense information for five open reading frames (ORFs). From 5' to 3'on the antigenome, the ORFs are p40, p23, gp18, p57 and pol. Proteinsp40, p23 and gp18 have been identified in infected cells and tissues:p40 and p23 are expressed at high levels in vitro and in vivo and arefound in the nucleus and cytoplasm of infected cells {Bause-Niedrig, I.,M. et al., Vet. Immunol. Immunopathol., 31:361-369 (1992)}. gp 18 is amembrane-associated glycoprotein that is expressed at lower levels. gp18was characterized in Example 2 below.

Messenger RNAs {Kliche, S., et al., J. Virol., 68: 6918-6923 (1994);Lipkin, W. I., et al., Proc. Natl. Acad. Sci. USA, 87:4184-4188 (1990);McClure, M. A., et al., J. Virol., 66:6572-6577 (1992); Pyper, J. M., etal., Virology, 195:229-238 (1993); Thibault, K. J., M.S. thesis;University of California, Irvine (1992); Thierer, J., et al., J. Gen.Virol., 73:413-416 (1992) and VandeWoude, S., et al., Science,250:1278-1281 (1990)} and proteins {Bause-Niedrig, I., et al., Vet.Immunol. Immunopathol., 31:361-369 (1992); Haas, B., et al., J. Gen.Virol., 67:235-241 (1986); Ludwig, H., et al., Progr. Med. Virol.,35:107-151 (1988); Schadler, R., et al., J. Gen. Virol., 66:2479-2484(1985) and Thiedemann, N., et al., J. Gen. Virol., 73:1057-1064 (1992)}corresponding to three of these ORFs, p40, p23 and gp18, have been foundin infected cells and tissues in a 5'-3' expression gradient(p40>p23>gp18) {Briese, T., et al., Proc. Natl. Acad. Sci. USA:91:4362-4366 (1994); Cubitt, B., et al., J. Virol., 68:1382-1396 (1994);and Richt, J. A., et al., J. Gen. Virol., 72:2251-2255 (1991)}.

Though Cubitt, B., et al., J Virol., 68:1382-1396 (1994) purported tohave sequenced the BDV genome, their paper contains numerous errors. Theerrors included (1) failure to recognize deletions in subgenomic RNAsdue to splicing; (2) misplacement of ORFs leading to the prediction of a40 kD protein instead of a 57 kD protein and failure to detect ORFoverlap of p57 with gp18 and pol; and (3) selection of incorrect motifsfor initiation of transcription. These mistakes were implicitlyacknowledged in a subsequent paper, de la Torre, J. C., J. Virol.,68:7669-7675 (1994). FIG. 1 of the latter paper incorporated the correctgenomic organization and transcription map described in Example 1 ofthis application. A later minireview which compares the sequencedifferences between the above Cubitt, et al.'s genomic sequence and thesequence described in Example 1 below concludes that the differencesseem most likely due to cloning and/or sequencing errors (of Cubitt etal.'s) rather than natural differences between the nucleotide sequencesof different strains. Schneeman, A., et al., to be published inVirology, 209 (1995); a co-author of the paper is Dr. Robert A. Lamb,the editor-in-chief of Virology and a Howard Hughes Medical InstituteInvestigator. Dr. Lamb was not a collaborator in the work described inExample 1 below..

In this Example, the 8,910 nucleotide BDV viral genome was cloned andsequenced using RNA from BDV particles. The viral genome hascomplementary 3' and 5' termini and contains antisense information forfive open reading frames. Homology to Filo-, Paramyxo- and Rhabdoviridaeis found in both cistronic and extracistronic regions. Northern analysisindicates that the virus transcribes mono- and polycistronic RNAs anduses termination/polyadenylation signals reminiscent of those observedin other negative-strand RNA viruses. BDV is likely to represent apreviously unrecognized genus, bornaviruses, or family, Bornaviridae,within the order Mononegavirales.

MATERIALS AND METHODS BDV cDNA Library Preparation and Screening

Genomic RNA template for library construction was obtained from anoligodendrocyte cell line (Oligo/TL) acutely infected with BDV Strain V{Briese, T., et al., Proc. Natl. Acad. Sci. USA 89:11486-11489 (1992)}.For the first genomic library, RNA from one viral particle preparationwas polyadenylated with poly(A) polymerase (GibcoBRL, Life Technologies,Inc., Grand Island, N.Y.) to facilitate cloning from the 3' terminus byoligo d(T) primed cDNA synthesis. Libraries were prepared in pSPORTusing the Superscript Plasmid system (GibcoBRL, Life Technologies, Inc.,Grand Island, N.Y.). The first library was screened using pAB5 and pAF4radiolabeled restriction fragments {Lipkin, W. I., et al., Proc. Natl.Acad. Sci. USA 87:4184-4188 (1990)}. Subsequent libraries were screenedusing radiolabeled restriction fragments from locations progressively 5'on the genomic RNA. 5'-terminal sequence from each library was used todesign an oligonucleotide primer for construction of the nextlibrary.DNA sequencing and sequence analysis. Plasmid DNA was sequencedon both strands by the dideoxynucleotide chain termination method{Sanger, F., et al., Proc. Natl. Acad. Sci. USA 74:5463-5467 (1977)}using bacteriophage T7 DNA polymerase (Sequenase version 2.0; UnitedStates Biochemical, Cleveland, Ohio). Five to ten independent clonesfrom each library were sequenced with overlap so that each region of thegenomic RNA was covered by at least two clones. Four libraries wereanalyzed yielding ≈8.9 kb of continuous sequence. Nucleic acid sequencewas analyzed using the Sequence Analysis Software Package (GeneticsComputer, Inc., Madison, Wis.). Database searches for related sequencesand multiple sequence alignments were performed using FastA and Pileup.

Sequence Determination at the 3' and 5' Termini of BDV Genomic RNA.Genomic RNA from one viral particle preparation (1-2×10⁸ cells) wastreated with tobacco acid pyrophosphatase (Epicentre Technologies,Madison, Wis.) and circularized with T4 RNA ligase (New England Biolabs,Inc., Beverly, Mass.) {Mandl, C. W., et al., BioTechniques 10:484-486(1991)}. The ligated RNA was reverse transcribed with Superscript II(Gibco BRL, Life Technologies, Inc., Grand Island, N.Y.) using primer5'-GCCTCCCCTTAGCGACACCCTGTA (SEQ ID NO: 11), complementary to a region465 nucleotides (nt) from the 5' terminus of the BDV genome. A 2 μlaliquot of the reverse transcription reaction was used to amplify theligated region by the polymerase chain reaction (PCR) using Stoffelfragment (Perkin-Elmer Cetus, Norwalk, Conn.). Primers used in the firstround of PCR were 5'-GCCTCCCCTTAGCGACACCCTGTA (SEQ ID NO: 11) and5'-GAAACATATCGCGCCGTGCA (SEQ ID NO: 12), located 241 nt from the 3'terminus of the BDV genome. Amplified products were subjected to asecond round of PCR using a nested set of primers:5'-TACGTTGGAGTTGTTAGGAAGC (SEQ ID NO: 13), 251 nt from the 5' terminus,and 5'-GAGCTTAGGGAGGCTCGCTG (SEQ ID NO: 14), 120 nt from the 3'terminus. PCR products were cloned {Schneider, P. A., et al., J. Virol.68:63-68 (1994)} and sequence across the 5'/3' junction was determinedfrom five independent isolates.

Northern hybridization. Poly(A)⁺ enriched RNA extracted from acutelyinfected rat brain using FastTrack (Invitrogen Corp., San Diego, Calif.)was size-fractionated on 0.22 M formaldehyde/1.0% agarose gels {Tsang,S. S., et al., BioTechniques 14:380-381 (1993)}, transferred toZeta-Probe GT nylon membranes (Bio-Rad Laboratories, Richmond, Calif.)and hybridized with random-primed 32P-labeled restriction fragments{Feinberg, A. P., et al., Anal. Biochem. 132:6-13 (1983)} representingORFs across the BDV genome (FIG. 6b). RNA transfer, hybridization andwashing were performed following the manufacturer's protocol (Bio-RadLaboratories, Richmond, Calif.).

RESULTS

The following figures present some of the results:

FIG. 3. (a) Organization of the BDV genome. Hatched boxes representcoding sequence complementary to ORFs for identified proteins, p40, p23,gp18, or putative proteins, p57, p180. (p180 is also referred to aspol.) Overlap is indicated by cross-hatched areas. Length of codingsequence corresponding to ORFs in nucleotides is indicated in brackets.Underlined italic numbers indicate length of sequence from stop codoncomplement to last templated uridine of termination/polyadenylylationsignal (black boxes). Italics with arrow indicate number of nucleotidesin intervening sequence between p40 polyadenylylation signal and p23coding sequence and between p23 polyadenylylation signal and gp18 codingsequence, respectively. Italics with dashed arrow indicate number ofnoncoding nucleotides at termini of the genome. (b) Coding potential ofgenome. Genomic sequence was translated in all six possible readingframes (3'-5' negative sense; 5'-3' positive sense) by using FRAMES(Genetics Computer Group). ORFs are indicated by bars and hatched boxes.

FIG. 4. Alignment of the p180 (pol) ORF and negative-strand RNA virusL-polymerase amino acid sequences with PILEUP. Solid lines indicateconserved L-polymerase motifs (a, A, B, C, D). BDV sequence (amino acid377 to 829 of SEQ ID NO:10) is indicated with double arrowheads.Rhabdoviridae: RaV, rabies virus (SEQ ID NO:33); VSV, vesicularstomatitis virus (SEQ ID NO:34); SYN, sonchus yellow net virus (SEQ IDNO:35). Paramyxoviridae: MeV, measles virus (SEQ ID NO:36); SeV, Sendaivirus (SEQ ID NO:37); NDV, Newcastle disease virus (SEQ ID NO:38); RSV,respiratory syncytial virus (SEQ ID NO:40). Filoviridae: MaV, Marburgvirus (SEQ ID NO:39). Numbers indicate amino acid range shown. Uppercaseletters in viral sequence lines indicate residues conserved in more thansix sequences. Uppercase letters in consensus line (Con) indicatepresence of identical or conserved amino acids in BDV. Agreement of BDVsequence with either rhabdo- or paramyxoviruses is indicated by * or x,respectively. +, Nonconserved glycine residue in BDV.

FIG. 5. Sequence analysis of BDV genomic termini. (a) Similarity of3'-terminal BDV sequence to leader regions of Rhabdoviridae: RaV (SEQ IDNO:47) and VSV (SEQ ID NO:48); Paramyxoviridae: MeV (SEQ ID NO:46), SeV(SEQ ID NO:44), NDV (SEQ ID NO:45), and RSV (SEQ ID NO:41); andFiloviridae: MaV (SEQ ID NO:43) and Ebola virus (EboV, SEQ ID NO:42).Abbreviations are as in FIG. 2. EboV, Ebola virus. Sequences are alignedby using arbitrary gap insertion to optimize nucleotide matching. (b)Comparison of complementarity at 3' and 5' termini of BDV genomic RNAwith that of four other nonsegmented, negative-strand RNA viruses. The3' and 5' terminal sequences for each virus are shown in viral RNA(3'-5', negative sense) orientation. Underlined sequence refers totranscriptional start of first gene or end of the L-polymerase gene(also referred to as "pol gene"), respectively (predicted for BDV). Theend of the L-polymerase gene of RaV is located outside the region shown.

FIG. 6. Map of BDV subgenomic RNAs relative to the viral antigenome. (a)Northern hybridization analysis of rat brain poly(A)⁺ RNA. Each lane washybridized with a probe representing a major BDV ORF as indicated by theletters A-E (see b). Results of hybridization with probes C* and E* wereidentical to results of hybridization with probes C and E, respectively(data not shown). Numbers at left indicate size of RNA markers inkilobases. Numbers at right indicate estimated size of majortranscripts. (b) Position of viral transcripts with respect toantigenome as determined by Northern hybridization and sequenceanalysis. Dashed lines indicate regions in the 1.5-kb RNA and the 6.1-kbRNA that contain a deletion. The boundaries of the deletions are notknown. Relative positions of probes used for Northern hybridization areshown. On the ORF map, potential start codons are indicated with upwardlines; ⋄, start codons predicted to be functional; x, potential startcodon present in strain V that is absent in strain He/80 (see text).Potential termination sites are indicated with downward lines. Use of T2and T3 has been confirmed (McClure, M. et al., J. Virol., 66:6572-6577;Thierer, J. et al., J. Gen. Virol., 73:413-416); use of T5 and T7 isconsistent with hybridization results. Termination at t1, t4 and t6 hasnot been observed (see a). (c) Alignment of the seven potentialtermination sites of BDV. Location of sites is indicated in the ORF map.Stop codons are underlined. Lowercase letters indicatetermination/polyadenylylation consensus sequence. Notermination/polyadenylylation site was found at or near the end of thegp18 ORF.

Sequencing of Genomic BDV RNA

Beginning from the 3' terminus, a series of four overlapping cDNAlibraries was constructed using BDV particle RNA {Briese, T., et al.,Proc. Natl. Acad. Sci. USA 89:11486-11489 (1992)} as template. Previousstudies have shown that the genomic RNA is not polyadenylated {de laTorre, J., et al., Virology 179:853-856 (1990)}. Thus, to construct thefirst library, genomic RNA was polyadenylated in vitro in order tofacilitate oligo d(T)-primed cDNA synthesis. For the subsequent threelibraries, genome-complementary oligonucleotide primers were designedbased on 5' terminal sequence determined in the previous round ofcloning. Each region of the genome was sequenced using a minimum of twoindependent clones. To determine the sequences at the termini, genomicRNA was circularized and sequenced across the junction using fiveindependent clones.

The 8,910 nt BDV genome contained antisense information for five majorORFs flanked by 53 nt of noncoding sequence at the 3' terminus and 91 ntof noncoding sequence at the 5' terminus (FIG. 3). In 3'-5' order, thefirst two ORFs encoded two previously described viral proteins, p40{McClure, M. A., et al., J. Virol. 66:6572-6577 (1992)} and p23{Thierer, J., et al., J. Gen. Virol. 73:413-416 (1992)}. The third,fourth and fifth ORF had coding capacities of 16 kDa (gp18), 57 kDa(p57) and 190 kDa (p180), respectively (FIG. 3a). Note: p180 is nowknown as "pol". Predicted amino acid sequence for the 16 kDa ORFcorrelated with microsequence data for an 18 kDa BDV glycoprotein (seethe section below for gp18 glycoprotein), originally described as theBorna disease-associated 14.5 kDa protein {Schadler, R., et al., J. Gen.Virol. 66:2479-2484 (1985)}. The first three ORFs showed no overlap andwere in frame with the fifth ORF (FIG. 3b). The 57 kDa ORF was in a+1/-2 frame relative to the other four ORFs and overlapped the adjacentORF for gp18 by 28 amino acids and ORF p180 by 34 amino acids. All ORFswere located on the (+) strand, complementary to the genomic RNA. ORFanalysis of the genomic (-) strand showed only three small ORF's, eachwith a coding capacity of less than 16 kDa (FIG. 3b).

Homology Analysis of Coding Sequence

Predicted amino acid sequence for the identified ORFs was used toexamine databases for similarity to other proteins. Previous analysis ofthe ORF encoding p40 had revealed distant sequence similarity toL-proteins of Paramyxoviridae and Rhabdoviridae {McClure, M. A., et al.,J. Virol. 66:6572-6577 (1992)}. FastA analysis of translated sequencefrom ORFs p23, gp18 and p57 showed no apparent similarity to other viralsequences; however, ORF p180 sequence consistently retrievedL-polymerases of Paramyxo- and Rhabdoviridae. Alignment of ORF p180(pol) sequence with sequence of RNA-dependent RNA polymerases ofnegative-strand RNA viruses showed conservation of both sequence andlinear order of regions homologous among these proteins. Extensiveconservation was found in the four characteristic motifs forL-polymerases of negative-strand RNA viruses (A-D in FIG. 4) {Poch, O.,et al., EMBO J. 8:3867-3874 (1989) and Poch, O., et al., J. Gen. Virol.71:1153-1162 (1990)}. With the exception of the glycine residue in motifB (position 322 of the alignment), conservation was found for theindividual amino acid residues postulated to participate in polymerasefunction {Poch, O., et al., EMBO J. 8:3867-3874 (1989)}. Conservationwas also found for a motif (a in FIG. 4) proposed to participate intemplate recognition {Poch, O., et al., J. Gen. Virol. 71:1153-1162(1990) and Barik, S., et al., Virology 175:332-337 (1990)}. TheGCG/pileup alignment placed ORF p180 sequence between polymerases ofParamyxo- and Rhabdoviridae. This intermediate position is reflected bythe presence of conserved amino acids which are in agreement with eitherthe rhabdo- or the paramyxovirus sequences (* or x, respectively; FIG.4). The distance between conserved motifs a and A was found to be shortin BDV as it is in rhabdoviruses, whereas this region is highly variablein length and sequence among paramyxoviruses {Poch, O., et al., J. Gen.Virol. 71:1153-1162 (1990)}. The GCG/pileup generated dendrogram,obtained using complete ORF p180 and L-protein sequences, indicated thatthe putative BDV polymerase was more closely related to L-polymerases ofRhabdoviridae than Paramyxoviridae.

Analysis of Noncoding Sequence at the Genomic Termini

3' terminal genomic sequence had a high A/U content of 60.5% with an Ato U ratio of ≈1:2, similar to 3' leader sequences of othernegative-strand RNA viruses. At the extreme 3' end, filo-, paramyxo- andrhabdoviruses have a common G/U rich region (FIG. 5a). In BDV, as inrespiratory syncitial virus, rabies virus and filoviruses, this regionwas not located at the 3' extremity. Comparison of the 3' and 5' terminiof BDV genomic RNA revealed complementarity similar to that found inother negative-strand RNA viruses {Keene, J. D., et al., J. Virol.32:167-174 (1979) and Tordo, N., et al., Virology 165:565-576 (1988)}(FIG. 5b). Alignment of the genomic termini allowed formation of aterminal panhandle, with the first three nucleotides unpaired. Thesubsequent complementary area of 6 nucleotides (positions 4-9 and8907-8902) could be extended by one gap insertion between position8901/8,902 resulting in an additional 10 nt stretch of complementaritywith a single mismatch (positions 18 and 8994; FIG. 5b).

Identification of Potential Termination/Polyadenylation Sites

Sequence preceding the poly(A) tracts of two cloned BDV mRNAs (UA₅){McClure, M. A., et al., J. Virol. 66:6572-6577 (1992) and Thierer, J.,et al., J. Gen. Virol. 73:413-416 (1992)} was used to analyze genomicsequence for homologous sites that could serve as potentialtermination/polyadenylation signals. Seven sites were found (FIG. 6c).Northern hybridization experiments supported use of four of these sites(T2, T3, T5 and T7) and allowed identification of atermination/polyadenylation signal consensus sequence (CMNMYYMNWA₆) (SEQID NO:57), where M is A or C, Y is C or U, and W is A or U. Only one ofthe three remaining sites (t6) matched the consensus sequence (FIG. 6c).

Northern Hybridization Analysis

Restriction fragments representing the five ORFs were used as probes forhybridization to poly(A)⁺ enriched RNA isolated from acutely infectedrat brain by FastTrack (FIGS. 6a and b). Because this procedure does notentirely eliminate poly(A)⁻ RNAs, small levels of BDV genome-size RNAcan usually be detected in these preparations. To allow determination ofthe relative abundance of RNAs detected by each probe, exposure timeswere normalized to the signal of the 8.9-kb RNA. Consistent with the 3'to 5' transcriptional gradient found for other negative-strand RNAviruses, of the eight subgenomic RNAs identified, those detected by the3'-most probes (genomic orientation), A and B, were more abundant thanthose detected by the more 5' probes (FIGS. 6a and b).

Mapping of the eight transcripts to the genome by Northern hybridizationindicated use of only three sites for transcriptional initiation andfour sites for termination. Probes C* and E* were used to distinguishbetween termination at T5 or t6 (FIG. 6b). The patterns of hybridizationwith probes C* and E* were identical to those obtained with probes C andE, respectively indicating termination at T5 (data not shown). Probescorresponding to p40 (A) and p23 (B) detected monocistronic RNAs of 1.2kb and 0.75 kb, respectively (FIG. 6). Probes A and B also detected a1.9 kb RNA consistent with failure of transcriptional termination at thep40 termination site {Pyper, J. M., et al., Virology 195:229-238(1993)}. Transcriptional readthrough was also found for polycistronictranscripts of 3.5, 2.8 kb and 7.1 kb. The 3.5 kb RNA detected by probesB, C, D and C*, is likely to initiate at or near the beginning of ORFp23 and terminate at T5. The 2.8 kb RNA detected by probes C, D and C*,is likely to initiate at or near the beginning of ORF gp18 and terminateat T5. The 7.1 kb detected by probes C, D, C*, E* and E, is likely toinitiate at or near the beginning of ORF gp18 and to continue through T5until it terminates at T7. Probes C and C* both hybridized to a 1.5 kbRNA and a 6.1 kb RNA. Interestingly, neither the 1.5 kb RNA nor the 6.1kb RNAs was detected by probe D, located between C and C* on the viralgenome. These findings are consistent with posttranscriptionalmodification resulting in a 1-1.3 kb deletion (FIG. 6).

DISCUSSION

The order Mononegavirales, which incorporates the families Filoviridae,Paramyxoviridae and Rhabdoviridae, has distinct characteristics thatinclude: (1) a nonsegmented negative sense RNA genome, (2) linear genomeorganization in the order 3' untranslated region/core proteingenes/envelope protein genes/polymerase gene/untranslated 5' region, (3)a virion associated RNA-dependent RNA polymerase, (4) a helicalnucleocapsid that serves as template for replication and transcription,(5) transcription of 5-10 discrete, unprocessed mRNAs by sequentialinterrupted synthesis from a single promoter and (6) replication bysynthesis of a positive sense antigenome {Pringle, C. R., et al., Arch.Virol. 117:137-140 (1991)}. The genomes of rhabdo-, paramyxo- andfiloviruses range in size from 11 to 20 kb. The BDV genome has beenestimated to be between 8.5 {Lipkin, W. I., et al., Proc. Natl. Acad.Sci. USA 87:4184-4188 (1990) and de la Torre, J., et al., Virology179:853-856 (1990)} and 10.5 kb {VandeWoude, S., et al., Science250:1276-1281 (1990) and Richt, J., et al., J. Gen. Virol. 72:2251-2255(1991)} in length. Our data confirm that the BDV genome, at only 8910nt, is smaller than those of other negative-strand RNA viruses. Severalfeatures suggest that BDV is a member of the order Mononegavirales:organization of ORFs on the genome, extensive sequence similarities ofthe largest BDV ORF to L-polymerases of rhabdo-, paramyxo- andfiloviruses, homology of 3' noncoding sequence to leader sequences ofMononegavirales and complementarity of BDV genomic termini.

In 5' to 3' antigenomic orientation, the first ORF contains 1110 nt. Dueto a more favorable translation initiation context {Kozak, M., NucleicAcids Res. 15:8125-8148 (1987)}, it is likely that the second AUG codon,39 nt inside the ORF, is used to express a 357 aa protein of 39.5 kDa(p40) {Pyper, J. M., et al., Virology 195:229-238 (1993)}. 26 ntdownstream of the stop codon is a polyadenylation signal {McClure, M.A., et al., J. Virol. 66:6572-6577 (1992)} (T2, FIGS. 6b and c). Thesecond ORF starts 79 nt from the p40 polyadenylation site. It has alength of 603 nt coding for a 201 aa protein of 22.5 kDa (p23). The stopcodon of ORP p23 is part of the polyadenylation signal {Thierer, J., etal., J. Gen. Virol. 73:413-416 (1992)} (T3, FIGS. 6b and c). Analysis ofthe intergenic region between ORFs p40 and p23 has shown that thissequence is less conserved among different BDV isolates than codingsequences for p40 and p23 {Schneider, P. A., et al., J. Virol. 68:63-68(1994)}. Therefore, expression of a small ORF in this region (x, FIG.3b); {VandeWoude, S., et al., Science 250:1276-1281 (1990) and Pyper, J.M., et al., Virology 195:229-238 (1993)} that overlaps with ORF p23seems unlikely {Schneider, P. A., et al., J. Virol. 68:63-68 (1994)}.Ten nt downstream of the p23 polyadenylation signal is the third ORF,426 nt in length, that codes for a 142 aa (16.2 kDa) protein. Due toglycosylation, the protein expressed from this ORF has a Mr of ≈18 kDa(gp18).

No polyadenylation signal similar to those identified for p40 and p23mRNAs {McClure, M. A., et al., J. Virol. 66:6572-6577 (1992) andThierer, J., et al., J. Gen. Virol. 73:413-416 (1992)} was found nearthe end of the gp18 ORF (FIGS. 6b and c). Instead, the following ORFoverlaps with the end of the gp18 ORF by 28 aa. It has a total size of1,509 nt that could code for a 503 aa protein of 56.7 kDa (p57). The ORFhas two AUG codons in the overlap with gp18. A third AUG located outsidethe overlap is 451 nt from the beginning of the ORF. Which, if any, ofthese AUGs is used is unknown as no protein has been identified. Apotential polyadenylation site is located 28 nt downstream of the p57ORF (t4). However, Northern hybridization results suggest that this siteis a weak or nonfunctional signal, because no major transcript(s) werefound to stop at this position (FIG. 6).

The fifth ORF encompasses more than half the length of the genome. Apotential polyadenylation site (T7), similar to that seen at the end ofORFs p40 and p23, is found 33 nt from the stop codon of p180 (pol)ORF(FIGS. 6b and c). Deletions identified by Northern hybridizationanalysis suggested that viral mRNAs might undergo post-transcriptionalmodification by RNA splicing. This hypothesis was subsequently confirmedby applicants {Schneider, P. A. et al., J. Virol., 68:5007-5012 (1994);Schneemann, A. et al. J. Virol., 68:6514-6522 (1994), herebyincorporated in their entirety.} RNA splicing extends the pol ORF by 459nucleotides allowing prediction of a protein of 190 kDa. {Schneider, P.et al., J. Virol., 68:5007-5012 (1994)}. Although functional studies ofBDV proteins have not yet been done, the organization of the viralgenome together with the limited biochemical data available suggestpossible roles for individual proteins in the virus life cycle. Fourlines of evidence suggest that p40 is likely to be a structural protein:(1) like nucleocapsid proteins (N) of rhabdo- and paramyxoviruses{Banerjee, A. K., et al., Pharmacol, Ther. 51:47-70 (1991)} (exceptpneumoviruses {Collins, P. L., The Paramyxoviruses, ed. Kingsbury, D. W.(Plenum, New York), pp. 103-162 (1991)}), p40 is found in the most 3'position on the genome; (2) p40 is similar in size to N proteins; (3)both p40 {Pyper, J. M., et al., Virology 195:229-238 (1993) and Ludwig,H., et al., Prog. Med. Virol. 35:107-151 (1988)} and N proteins{Banerjee, A. K., et al., Pharmacol, Ther. 51:47-70 (1991)} are abundantin infected cells and particles; (4) neither N proteins {Banerjee, A.K., et al., Pharmacol, Ther. 51:47-70 (1991)} nor p40 {Thiedemann, H.,et al., J. Gen. Virol. 73:1057-1064 (1992)} are phosphorylated orglycosylated. p23, a phosphorylated protein {Thiedemann, H., et al., J.Gen. Virol. 73:1057-1064 (1992)}, is in the next position on the genome.ORF p23 corresponds in position to genes coding for phosphoproteins inParamyxoviridae (P) and Rhabdoviridae (NS) {Banerjee, A. K., et al.,Pharmacol, Ther. 51:47-70 (1991)}. This suggests that p23 might serve asimilar role in the BDV system. In support of this hypothesis, GCGanalysis showed that the protein has a high Ser/Thr content (16%), ischarged (pI 4.8) and contains a N-terminal cluster of acidic amino acidscompatible with structural features of P/NS proteins {Banerjee, A. K.,et al., Pharmacol, Ther. 51:47-70 (1991)}. In previously describedMononegavirales, the next gene codes for matrix protein (M) {Banerjee,A. K., et al., Pharmacol, Ther. 51:47-70 (1991)}. gp18 occupies thisposition on the BDV genome. Though small for a matrix protein, gp18 hasa predicted pI, 10, that is close to the basic pI of M proteins, ≈9, andits membrane-association would be compatible with a matrix proteinfunction. For p57, computer analysis predicted similarities toglycoproteins of negative-strand RNA viruses: potential glycosylationsites as well as N-terminal and C-terminal hydrophobic "anchor" domains(data not shown). The largest ORF (pol) is located most 5' on thegenome. Its size, 5' position and conservation of motifs consideredcritical to L-polymerase activity, suggest that this ORF is likely tocode for the BDV polymerase (FIG. 6).

Analysis of Northern hybridization experiments in conjunction withgenomic sequence data has allowed construction of a tentativetranscription map (FIG. 6). While it has not been possible to identifysignals for initiation of transcription by using consensus sequences ofother negative-strand RNA viruses, we have identified consensus sequencefor termination/polyadenylation in BDV using known ends of p40 and p23mRNAs {McClure, M. A., et al., J. Virol. 66:6572-6577 (1992) andThierer, J., et al., J. Gen. Virol. 73:413-416 (1992)} (FIG. 6c). Thesesequences appear to function as weak termination signals. Unlike othernegative-strand RNA viruses, BDV shows a high frequency of readthroughtranscripts. Organization and sequence similarities to Filo-, Paramyxo-and Rhabdoviridae suggest that BDV is a member of the orderMononegavirales. Dependent on the parameters and regions selected forhomology analysis, BDV can be represented as being more closely relatedto filo-, paramyxo- or rhabdoviruses. Overlap of coding sequence, highfrequency of polycistronic readthrough transcripts andposttranscriptional modification are properties of the BDV system notfound in other members of the order Mononegavirales. These featurescould serve as independent mechanisms for modulation of gene expressionto achieve the persistent, non-cytopathic infection that is a cardinalcharacteristic of this neurotropic virus.

EXAMPLE 2 BDV Glycoprotein gp18

Using methods for isolation of the 14.5-kDa protein {Schadler, R., etal., J. Gen. Virol., 66:2479-2484 (1985)}, we have purified aglycoprotein from BDV-infected rat brain that is encoded by a429-nucleotide (nt) ORF located 3' to ORF p23 on the viral antigenome.The protein is predicted to be 16.2 kDa; glycosylation results in a 1-to 2-kDa increase in molecular weight. This glycoprotein, gp18, is thefirst glycoprotein to be identified in the BDV system. Lectin bindingand endoglycosidase sensitivity assays suggest that gp18 is an unusualN-linked glycoprotein.

MATERIALS AND METHODS Infection of Animals and Cultured Cells

Animals and cells were infected with BDV strain He/80 {Herzog, S., etal., Med. Microbiol. Immunol., 168:153-158 (1980) and Schneider, P. etal., Virol. 68:63-68 (1994)}. Newborn Lewis rats were infected byintracranial injection with 1.5×10⁴ focus-forming units of BDV. Threeweeks after infection, animals were sacrificed and brains were removedfor isolation of BDV particles {Carbone, K., et al., J. Virol.,61:3431-3440 (1987)} or gp18. C6 cells and MDCK cells were persistentlyinfected with BDV as described previously {Carbone, K. M., J. Virol.,67:1453-1460 (1993) and Herzog, S., et al., Med. Microbiol. Immunol.,168:153-158 (1980)}. Monolayers of rabbit fetal glial cells were acutelyinfected by adding BDV at 1.0 focus-forming unit per cell to the culturemedium (Dulbecco modified Eagle medium, 5% fetal calf serum; Gibco BRL,Grand Island, N.Y.).

Protein Purification and Microsequencing

Protein was purified from infected cells and tissues by detergent-saltextraction by the method of Schadler et al. {Schadler, R., et al., J.Gen. Virol., 66:2479-2484 (1985)}. For microsequencing, protein wascleaved with 10% cyanogen bromide in 75% formic acid (Sigma ChemicalCo., St. Louis, Mo.). Peptide fragments were separated by reverse-phasehigh-performance liquid chromatography (RP-HPLC) on a Vydac C-18 column,using a trifluoroacetic acidacetonitrile gradient. Sequencedeterminations were performed by automated Edman degradation on aHewlett-Packard model G1000A protein sequencer.

Antibodies

Antibodies to purified gp18 were produced in 3-month-old BALB/c mice.Animals were injected subcutaneously with 5 μg of protein in Freund'scomplete adjuvant and boosted 3 weeks later with a subcutaneousinjection of 3 μg of protein in Freund's incomplete adjuvant. For 6weeks thereafter, at 2-week intervals, animals received intraperitonealinjections of 5 μg of protein in phosphate-buffered saline (PBS) with 5μg of lipopolysaccharide (Salmonella typhimurium; Difco, Detroit, Mich.)(three injections). Blood was drawn every 2 weeks during weeks 7 through28 for measurement of serum antibody titer to purified protein byWestern blotting (immunoblotting). Antisera collected at week 28 wereused for virus neutralization studies. Rabbit antisera to recombinantBDV p40 and p23 were used as controls (see Example 3, below).

Cloning and Sequencing of cDNA Encoding gp18

gp18-specific oligonucleotides were used to amplify full-length codingsequence for gp18 from two BDV-infected adult rat brain cDNA libraries{Lipkin, W. I., et al., Proc. Natl. Acad. Sci. USA, 87:4184-4188 (1990)and McClure, M. A., et al., J. Virol. 66:6572-6577 (1992)} as well astotal cellular RNA {Chirgwin, J. J., et al., Biochemistry, 18:5294-5299(1979)} and poly(A)⁺ RNA {Aviv, H., et al., Proc. Natl. Acad. Sci. USA,69:1408-1412 (1972)} extracted from infected rat brain. Reversetranscription (RT) was performed with an oligo(dT) primer andSuperscript II (Gibco BRL, Life Technologies, Inc., Grand Island, N.Y.).PCR was carried out with Ampli-Taq Stoffel fragment according tostandard protocols (Perkin-Elmer, Norwalk, Conn.) with the followingprimer pair: 5'-terminal XhoI-gp18 sense oligonucleotide (XhoI-gp18-S1),TCCTCGAGATGAATTCAAAACATTCCTATC (nt 1892 to 1914; XhoI restriction siteindicated by underlining) (SEQ ID NO: 15); and 3'-terminal gp18antisense oligonucleotide (gp18-AS1), CTAAGGCCCTGAAGATCGAAT (nt 2301 to2321) (SEQ ID NO: 16). Products were purified by agarose gelelectrophoresis using a USBioclean purification kit (U.S. Biochemical,Cleveland, Ohio) and cloned into Bluescript SKII+ (Stratagene, SanDiego, Calif.) prepared with 3' T overhangs {Marchuk, D., et al.,Nucleic Acid Res., 19:1154 (1990)}. A minimum of three independentclones from each template source was sequenced on both strands by thedideoxynucleotide chain termination method using bacteriophage T7 DNApolymerase (Sequenase; U.S. Biochemical, Cleveland, Ohio). The plasmidresulting from amplification of neonatally infected rat brain RNA wasnamed pBDV-gp18.

In vitro Transcription, Translation, and Cotranslational Processing

Plasmid clones pBDV-gp18 and pBDV-23 {Thibault, K. J., M.S. thesis,University of California, Irvine (1992)} linearized with EcoRI were usedas templates for in vitro synthesis of capped RNA transcripts.Transcription products or Saccharomyces cerevisiae α-factor mRNA(control for glycosylation) were translated in vitro by usingnuclease-treated rabbit reticulocyte lysates (Promega Corp., Madison,Wis.) in the presence of [³⁵ S]methionine (Amersham Corporation,Arlington Heights, Ill.). Cotranslational processing was assessed by invitro translation using reticulocyte lysates supplemented with caninemicrosomal membranes (Promega, Madison, Wis.). Transcription,translation, and cotranslational processing studies were performedaccording to the manufacturer's protocols. Translation products wereimmunoprecipitated with mouse anti-gp18 serum and then size fractionatedby sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis(PAGE) (13% gel) {Laemmli, U. K., et al., J. Mol. Biol., 80:575-581(1973)} for autoradiographic analysis. Methods for immunoprecipitationand autoradiography have been described elsewhere {Lipkin, W. I., etal., Proc. Natl. Acad. Sci. USA, 87:4184-4188 (1990)}.

Protein Gel Electrophoresis and Immunoblotting

Proteins were size fractionated by SDS-PAGE (12% gel) and thentransferred to Immobilon-N membranes (Millipore Corp., Bedford, Mass.).Primary antisera for immunoblotting were from rats chronically infectedwith BDV (day 100 after intracranial infection) or mice immunized withpurified gp18. The secondary antibody was alkalinephosphatase-conjugated goat antimouse immunoglobulin G (Sigma ChemicalCo., St. Louis, Mo.); the substrate was Western Blue (Promega Corp.,Madison, Wis.).

Carbohydrate Analysis

Purified protein was size fractionated by SDS-PAGE (13% gel) and theneither silver stained for detection of protein or carbohydrate {Tsai, C.M., et al., Anal. Biochem., 119:115-119 (1982)} or transferred toImmobilon-N membranes (Millipore, Bedford, Mass.) for lectin staining.The carbohydrate composition of immobilized protein was determined byusing a DIG Glycan Differentiation Kit (Boehringer Mannheim,Indianapolis, Ind.) and peroxidase-labeled Bandeiraea simplicifoliaagglutinins I and II (BS-I and BS-II; Sigma Chemical Co., St. Louis,Mo.). The substrate for peroxidase was 4-chloro-1-naphthol (PierceChemical Company, Rockford, Ill.). Glycosidase digests of native anddenatured protein (incubated for 5 minutes at 100° C. in 0.01% SDS) wereperformed according to the manufacturer's protocols, using the followingendoglycosidases: endoglycosidase F and N-glycosidase F; O -glycosidase;N-glycosidase F; endoglycosidase F, N-glycosidase free; endoglycosidaseH; and endo-β-galactosidase (Boehringer Mannheim).

RESULTS

The following figures present some of the results:

FIG. 7. Sequence of ORF gp18. The diagram shows the location of ORF gp18on the viral antigenome (5'-3') relative to ORFs p40 and p23 (boxes).ORF gp18 sequences were from Oligo/TL cells infected with BDV strain V(SV) and rat brain infected with BDV He/80 (RB). Peptide sequences (P#1,P#2, and P#3) were obtained by microsequencing of purified protein fromHe/80-infected rat brain. Periods indicate identical nucleotide or aminoacid sequences. Variable amino acid residues (large asterisk) and stopcodons (small asterisks) are indicated. Underlining indicates potentialglycosylation sites.

FIG. 8. Glycan determination of gp18. gp18 isolated from infected ratbrain was size fractionated by SDS-PAGE (12% gel) then transferred to anImmobilon-N membrane for lectin staining (see Materials and Methods).Lanes: 0, protein detection by mouse anti-gp18 serum; 1, ConA; 2, wheatgerm agglutinin; 3, D. stramonium agglutinin; 4, BS-I; 5, BS-II; 6, G.nivalis agglutinin; 7, S. nigra agglutinin; 8, M. amrensis agglutinin;9, peanut agglutinin. Positions of molecular weight markers are shown inkilodaltons at the right.

FIG. 9. gp18 is sensitive to endoglycosidases. gp18 isolated frominfected rat brain was treated with either buffer alone orendoglycosidase. Protein was size fractionated by SDS-PAGE (13% gel) anddetected by silver staining. Lanes: 1, buffer; 2, endoglycosidase F plusN-glycosidase F; 3, endoglycosidase F (N-glycosidase free); 4,endo-β-galactosidase. Positions of molecular weight markers are shown inkilodaltons at the right.

FIG. 10. In vitro transcription, translation, and cotranslationalprocessing of gp18. RNA transcripts were synthesized from pBDV-23 (anonglycosylated BDV protein control) or pBDV-gp18 and translated invitro by using rabbit reticulocyte lysates in either the absence orpresence of canine microsomal membranes. [³⁵ S]methionine-labeledtranslation products were immunoprecipitated with antisera to p23 orgp18 and protein A-Sepharose and then size fractionated by SDS-PAGE (13%gel) for autoradiography (A) or transferred to Immobilon-N membranes forConA lectin staining (B). Translated gp18 in lane 5 of panel A and lane3 of panel B was incubated with endoglycosidase F plus N-glycosidase Fprior to SDS-PAGE. (A) Lanes: 1, pBDV-23 RNA; 2, pBDV-23 RNA plusmicrosomal membranes; 3, pBDV-gp18 RNA; 4, pBDV-gp18 RNA plus microsomalmembranes; 5, pBDV-gp18 RNA plus microsomal membranes, incubated withendoglycosidases. The long arrow indicates the position of glycosylatedprotein (lanes 3 and 4); the short arrow indicates the position ofprotein after treatment with endoglycosidase F plus N-glycosidase F(lane 5). The asterisk indicates nonspecific background signal (lane 5).Positions of molecular weight markers are shown in kilodaltons at theright. (B) Lanes: 1, pBDV-gp18 RNA; 2, pBDV-gp18 RNA plus microsomalmembranes; 3, pBDV-gp18 RNA plus microsomal membranes, incubated withendoglycosidases.

Isolation of gp18

Protein was isolated from neonatally infected rat brain, acutelyinfected rabbit fetal glial cells (two passages), persistently infectedC6 cells, and persistently infected MDCK cells, using the method ofSchadler et al. {Schadler, R., et al., J. Gen. Virol., 66:2479-2484(1985)}. The purity of the protein was confirmed by silver staining ofthe protein after SDS-PAGE (data not shown). The quantity of protein wasestimated in silver-stained gels by using lysozyme standards. Typicalyields were 5 μg of protein from one neonatally infected rat brain and 2μg of protein from 10⁸ infected cultured cells. Protein from neonatallyinfected rat brain was used for microsequencing, carbohydrate analysis,and immunization of mice.

Protein and Nucleic Acid Sequence Analysis

Direct microsequencing of gp18 was not possible because of a blockedamino terminus; thus, to allow analysis, the protein was cleaved withcyanogen bromide. Sequencing of the cleavage mixture indicated thepresence of three N termini. From the mixture, two peptides (peptides 1and 3; FIG. 7) were isolated by RP-HPLC and sequenced individually,allowing inference of a third sequence (peptide 2; FIG. 7) bysubtraction. Peptide sequences were used as probes to search ORFslocated on the BDV antigenome. The peptide sequences obtained from thepurified gp18 mapped to a 429-nt ORF (ORF gp18) on the viral antigenomethat predicts a 142-amino-acid protein with a molecular weight of 16,244(FIG. 7).

Genomic sequence corresponding to the gp18 ORF was used to design probesand primers for identifying mRNA encoding gp18. In each of two cDNAlibraries prepared from BDV-infected adult rat brain poly(A)⁺ RNA{Lipkin, W. I., et al., Proc. Natl. Acad. Sci. USA, 87:4184-4188 (1990)and McClure, M. A., et al., J. Virol., 66:6572-6577 (1992)}, 100,000recombinants were screened by hybridization with a 271-bp HincII-HinfIrestriction fragment from pTB-BDV 5.82 (nt 2062 to 2333 in the viralgenome) {Briese, T., et al., Proc. Natl. Acad. Sci. USA 91:4362-4366(1994)}. These libraries were also screened by PCR using the 5'-terminalXhoI-gp18 sense primer (nt 1892 to 1914) and oligo(dT). Total cellularand poly(A)⁺ RNAs extracted from persistently infected C6 cells,BDV-infected adult rat brain, or 3-week-old neonatally infected ratbrain (the peak time point for in vivo expression of gp18) weresubjected to RT-PCR using oligo(dT) in combination with the 5'-terminalXhoI-gp18 sense primer. No gp18-specific transcript corresponding to thesize of ORF gp18 was obtained in these experiments. In contrast, use ofthe 5'-terminal XhoI-gp18 sense primer in combination with a 3'-terminalgp18 antisense primer (nt 2301 to 2321) allowed amplification of gp18sequences from any of these sources by RT-PCR. In spite of variabilityat the nucleic acid level, the predicted amino acid sequence obtainedfrom the different sources was the same as for strain V genomicsequence, with the exception of a single exchange in position 108 (E→D)(FIG. 7).

Characterization of gp18 as a Glycoprotein

Purified gp18 was size fractionated by SDS-PAGE. Modified silverstaining revealed the presence of carbohydrate; thus, fractionatedprotein was blotted onto Immobilon-N membranes to determine the presenceof individual saccharides through lectin binding studies. Binding wasobserved with Cancanavalia ensiformis agglutinin (ConA), wheat germagglutinin, Datura stramonium agglutinin, BS-I, and BS-II but not withGalanthus nivalis agglutinin, Sambucus nigra agglutinin, Maackiaamurensis agglutinin, and peanut agglutinin (FIG. 8). This stainingpattern was consistent with the presence of N-acetylglucosamine,N-acetylgalactosamine, mannose, and galactose. In addition, native anddenatured proteins were digested with specific endoglycosidases, sizefractionated by SDS-PAGE, and then stained to assess molecular weightshift and presence or absence of carbohydrate. Treatment withO-glycosidase or endoglycosidase H had no effect (data not shown). Incontrast, treatment with endoglycosidase F and N-glycosidase F resultedin a loss of 1 to 2 kDa (FIG. 9) and abrogation of lectin staining withConA (data not shown). Treatment with endoglycosidase F (N-glycosidasefree) or endo-β-galactosidase also resulted in a loss of 1 to 2 kDa(FIG. 9).

In vitro Transcription, Translation, and Processing of gp18

With linearized pBDV-gp18 used as a template, gp18 RNA was transcribedand translated in vitro in either the presence or absence of caninemicrosomal membranes. The gp18 RNA directed translation of two proteinsof 16 and 18 kDa that were recognized by monospecific murine antiserumto purified gp18. Translation in the presence of microsomal membranesled to an increase in the relative proportion of the 18-kDa protein.Treatment with endoglycosidase F resulted in loss of the 18-kDa proteinspecies (FIG. 10A). Glycosylation of the 18-kDa species was also shownby lectin binding studies performed after translation products were sizefractionated by SDS-PAGE and transferred to membranes. The 18-kDaprotein was recognized by ConA, whereas the 16-kDa protein did not bindConA (FIG. 10B). Modification of translated protein by the microsomalmembranes was specific for gp18. Translation of RNA encoding BDV p23,which encodes a potential N-glycosylation site (amino acids 53 to 55),included as a negative control for in vitro glycosylation, was notinfluenced by the presence of microsomal membranes (FIG. 10A).

DISCUSSION

We have isolated and partially characterized a BDV glycoprotein withunusual properties. This protein, previously reported as 14.5 kDa{Schadler, R., et al., J. Gen. Virol., 66:2479-2484 (1985)}, is 16.2 kDaprior to carbohydrate modification and ≈18 kDa after glycosylation.Though no classical sites for N linkage (N-x-S/T {Marshall, R. D., Annu.Rev. Biochem, 41:673-702 (1972)}) are found in the gp18 sequence, theprotein is readily modified in vitro in the presence of a microsomalmembrane system capable of N glycosylation {Gahmberg, C. G., et al., p.281-297, In S. Fleischer and B. Fleischer (ed.), Biomembranes, AcademicPress, New York (1983) and Lau, J. T. Y., et al., J. Biol. Chem.,258:15255-15260 (1983)}. In addition, gp18 is sensitive to N-glycosidaseF, an enzyme which cleaves between asparagine and N-acetylglucosamine{Plummer, T. H., et al., J. Biol. Chem., 259:10700-10704 (1984) andTarentino, A. L., et al., Biochem., 24:4665-4671 (1985)}. These findingsindicate that gp18 is N glycosylated at a nonclassical site. Onepotential site is N-I-Y (amino acids 74 to 76). The presence of ahydroxyl amino acid (T or S) or cysteine in position +2 (N-x-T/S or C)has been proposed as essential for hydrogen bond donor function in Nglycosylation {Bause, E., et al., Biochem. J., 195:639-644 (1981)}. Itis possible that tyrosine (Y), another hydroxyl amino acid in position+2, could serve as a hydrogen bond donor in gp18. A second potentialsite for N glycosylation is L-N-S-L-S (amino acids 87 to 91 of SEQ IDNO:6), which is similar to S-N-S-G-phosphorylated S (SEQ ID NO:59), thesite for N glycosylation in a glycopeptide from hen yolk phosvitin{Shainkin, R., et al., J. Biol. Chem., 246:2278-2284 (1971)}.

gp18 is sensitive to endoglycosidase F, an enzyme that cleaves after theN-linked N-acetylglucosamine in high mannose-, biantennary hybrid-, andbiantennary complex-type oligosaccharides {Tarentino, A. L., et al.,Biochem., 24:4665-4671 (1985) and Tarentino, A. L., et al., MethodsEnzymol., 230:44-57 (1994)}. The protein is not sensitive toendoglycosidase H, an enzyme which cleaves after the N-linkedN-acetylglucosamine in high-mannose- and most hybrid-typeoligosaccharides but does not cleave complex-type oligosaccharides{Trimbel, R. B., et al., Anal Biochem., 141:515-522 (1984)}. Lectinstaining using G. nivalis agglutinin shows no evidence of terminalmannose characteristic for hybrid- and high-mannose-type glycosylation.In contrast, staining with ConA (mannose, N-acetylglucosamine, branchedtrimannosyl core) {Ogata, S., et al., J. Biochem., 78:687-696 (1975)},wheat germ agglutinin (N-acetylglucosamine), and BS-II (terminalN-acetylglucosamine) {Ebisu, S., et al., Methods Enzymol., 50:350-354(1978)} indicates the presence of terminal N-acetylglucosamine andinternal mannose. Thus, there is evidence from the pattern ofendoglycosidase sensitivity and lectin staining that gp18 is likely tobe a biantennary complex-type glycoprotein.

gp18 is sensitive to endo-β-galactosidase. This enzyme cleaves betweengalactose and either N-acetylglucosamine or galactose when thesesaccharides occur in unbranched sequence {Scudder, P., et al., J. Biol.Chem., 259:6586-6592 (1984)}. The presence of galactose was confirmed byBS-I lectin binding (FIG. 8). The presence of both N-acetylglucosamineand galactose was confirmed by high-performance anion-exchangechromatography with pulsed amperometric detection. The combination ofN-acetylgalactosamine and galactose is usually found in O-linkedcarbohydrates {Hayes, B. K., et al., J. Biol. Chem. 268:16170-16178(1993)}. Though it is possible that gp18 is both N and O glycosylated,N-acetylgalactosamine has also been reported to occur in complex-typeN-linked glycosylation {Hayes, B. K., et al., J. Biol. Chem.268:16170-16178 (1993)}.

We did not detect a monocistronic ≈429-nt mRNA for gp18 by PCR usingoligo(dT), a 5' sense primer, and template from a variety of sources,including infected cell lines and rat brain. In contrast, a 429-nt gp18cDNA was readily amplified by using gene-specific primers and total RNAor poly(A)⁺ RNA as a template. Northern (RNA) hybridization experimentswith gp18-specific probes using total RNA or poly(A)⁺ RNA from infectedcells or rat brain detected only 1.5-, 2.8-, 3.5-, 6.1-, and 7.1-kbtranscripts. Recent experiments confirmed that the 1.5- and 2.8-kb RNAscan serve as templates for in vitro translation of the gp18 (data notshown). These data suggest that gp18 is likely to be translated from oneor more of the larger RNA transcripts.

The role of gp18 in the BDV life cycle remains to be determined. Thoughthe virus has not been characterized morphologically, genetic analysishas characterized BDV as a member of the order Mononegavirales {Briese,T., et al., Proc. Natl. Acad. Sci. USA 91:4362-4366 (1994) and Cubitt,B., et al., J. Virol., 68:1382-1996 (1994)}. In nonsegmented,negative-strand RNA viruses, the third gene usually directs expressionof a matrix protein. Matrix proteins in members of the orderMononegavirales are not known to be glycosylated; however, glycosylatedmatrix proteins that resemble gp18 in size and pI (≈10) have been foundin other viral systems (e.g., E1 in coronaviruses {Armstrong, J., etal., Nature (London), 308:751-752 (1984)}). Preliminary observationssuggest that gp18 is present on the surface of the viral envelope.Monospecific antisera and monoclonal antibodies to gp18 precipitatedviral particles and had neutralizing activity. In contrast, antibodiesto p40 and p23 did not precipitate viral particles or neutralizeinfectivity (see Example 4 below). Preincubation of primary rabbit fetalglia (cells highly susceptible to BDV) with gp18 prevented infection. Nosuch effect was observed with either p40 or p23. Last, gp18 and BDVparticles compete for binding to a ≈100-kDa membrane protein present incells susceptible to infection.

Expression of Recombinant P57

cDNAs representing the p57 ORF were amplified by RT-PCR using BDV(strain He/80)-rat brain RNA as template. The amplified p57 cDNA wassubcloned into two plasmid vectors, pET21b (Novagen) and pSFV-1 (GIBCOBRL).

pET21b, a prokaryotic expression vector, was selected because it allowsfor tight control of protein expression, an important feature forexpression of proteins toxic to host cells. The N-terminus of p57contains a hydrophobic sequence that confers extreme toxicity toprokaryotic cells. Therefore, to facilitate the expression of p57, thefirst 152 N-terminal amino acids were excluded during the cloning. PCRamplified cDNA representing nucleotides 2697 to 3743 of p57 ORF (aminoacids 153 to 503) was generated by using oligonucleotide primersdesigned with a 5' restriction site (BamHl for sense primer; Xhol forantisense primer). The PCR product was cloned into pET21b at the BamHland Xhol restriction sites, thus generating pET21b-BDV57₁₅₃₋₅₀₃. ThepET21b-BDV57₁₅₃₋₅₀₃ plasmid was transformed into BL21 host cells andrecombinant protein was expressed and purified by using protocolsprovided by the manufacturer.

An eukaryotic expression system, which allows for posttranslationalmodification, was selected for the expression of a recombinant proteinmore similar to native p57. pSFV-1 is a eukaryotic expression vectorthat can be used to generate a replication defective Semliki Forestvirus (SFV) genomic RNA. The entire p57 ORF was PCR amplified and clonedinto pSFV-1 prepared with 3' T-overhangs at the Smal site, thusgenerating pSFV-BDV57. Transfection of pSFV-BDV57 transcripts intomammalian cells, results in overexpression of the posttranslationallyprocessed p57 gene product.

EXAMPLE 3 ELISA for the Detection of Antibodies to Borna Disease VirusProteins

We have expressed p40, p23 and gp18 as recombinant proteins andestablished a sensitive, specific ELISA for analyzing immunoreactivityto BDV. This assay system is more sensitive and rapid than methodscurrently employed for serologic diagnosis of infection such as Westernblot, indirect immunofluorescent test (IFT) or immunoprecipitation.

This system provides a convenient tool for diagnosing disease,determining the prevalence of infection in animal and human populationsand mapping the antigenic determinants for the immune response ininfected hosts.

MATERIALS AND METHODS Infection of Animals and Cultured Cells

Six week old Lewis rats (Charles River) were infected intranasally with6×10⁴ focus forming units (ffu) of BDV strain He/80-1 {Carbone, K., etal., J. Virol., 61:3431-3440 (1987) and Schneider, P. A., et al., J.Virol., 68:63-68 (1994)}. C6 cells were persistently infected with BDVHe/80-1 (C6BDV) {Carbone, K. M., et al., J. Virol., 67:1453-1460(1993)}. Rabbit fetal glial cells were infected with BDV He/80-1 at amultiplicity of one ffu per cell then passaged once before use in IFTassays. BDV strain He/80 was originally isolated from infected horsebrain, passaged twice in rabbits, three times in rabbit fetal glialcells, and twice in Lewis rats {Herzog, S., et al., Med. Microbiol.Immunol., 168:153-158 (1980)}. He/80-1 was passaged four additionaltimes in Lewis rats and used for infection of animals and cell lines.

Generation of Recombinant Proteins (recp40, recp23, and recp18)

Full length cDNAs encoding p40, p23 or gp18 were cloned into theprokaryotic expression vector pET15b (Novagen) for production ofrecombinant proteins. pBDV-40 in pcDNA II {McClure, M. A., et al., J.Virol., 66:6572-6577 (1992)} was amplified using the primers p40Xho I(5'-CCCTCGAGGACCAAGATTT-3') (SEQ ID NO: 17) and Sp6 (20mer, PromegaCorp., Madison, Wis.). pBDV-23 in pBluescript SKII+ {Thibault, K. J.,M.S. thesis, University of California, Irvine (1992)} was amplified withthe primers p24Nde I (5'-AGAATCATATGGCAACGCGACCATC-3') (SEQ ID NO: 18)and T7 (20mer Promega). Polymerase chain reaction was performed usingTaq polymerase (Perkin-Elmer Cetus Corp., Norfolk, Conn.) according tothe manufacturer's protocol. Products amplified from pBDV-40 and pBDV-23were phenol/chloroform extracted, precipitated and digested with BamH Iand either Xho I (pBDV-40) or Nde I (pBDV-23) (Promega Corp., Madison,Wis.). pBDV-cp18 in pBluescript SKII+ (see Example 2 above) was digestedwith Xho I and BamH I. Digested fragments were purified by agarose gelelectrophoresis (USB, USBioclean, Cleveland, Ohio) and cloned intopET15b (Novagen Corporation, Madison, Wis.). Protein expression inplasmid containing Escherischia coli cells was induced by addition ofisopropyl-β-thiogalactopyranoside (1 mM) for 3 hours at 37° C. Proteins(recp40, recp23, and recp18) were purified by nickel-chelate affinitychromatography according to manufacturer's instructions (Novagen Corp.).Purification was assessed by SDS-PAGE and antigenicity was confirmed byWestern blot using sera from infected rats. Proteins were dialyzedagainst 150 mM NaCl and 2.5 mM CaCl₂ and digested with biotinylatedthrombin (1 unit/mg recombinant protein, Novagen Corp.) overnight atroom temperature. Thrombin was removed using streptavidin-agarose(Novagen Corp.) according to manufacturer's protocol. Proteinconcentrations were estimated by BioRad protein assay according tomanufacturer's instructions.

Antibodies to BDV and Recombinant BDVproteins

Sera were collected from infected rats at time of sacrifice or by tailbleeding at 2-week intervals after inoculation with BDV. Antibodies torecp40 and recp23 were each produced in two rabbits. Animals wereinjected subcutaneously (s.c.) with 25 μg of protein in Freund'scomplete adjuvant and then boosted 3 weeks later s.c. with 25 μg ofprotein in Freund's incomplete adjuvant. After 6 weeks some animalsreceived an additional s.c. injection of 25 μg protein in Freund'sincomplete adjuvant. Blood was collected at 2-week intervals duringweeks 7 through 14 for detection of antibodies by Western blot andELISA.

Indirect Immunofluorescent Test (IFT)

Rabbit fetal glial cells were processed for titration of serumantibodies against BDV using the immunohistochemical methods of Pauli etal. {Pauli, G., et al., Zbl. Vet. Med. [B] 31:552-557 (1984)}. Briefly,infected and noninfected cells were fixed with 4% formaldehyde in PBS,permeabilized with 1% Triton X-100 in PBS and blocked with 1% fetalbovine serum (FBS) in PBS. After incubation with sera diluted in 1% FBSin PBS, cells were incubated with fluorescein-conjugated goat anti-ratIgG and IgM or goat anti-rabbit IgG (Sigma Chemical Co., St. Louis, Mo.)diluted 1:200 in 1% FBS in PBS and then examined by fluorescentmicroscopy. The IFT titer for each serum was determined to be theendpoint dilution at which specific inununoflourescence was detected.

Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE),Western Blot (WB) and Immunoprecipitation (IP)

For WB, lysates from infected and noninfected C6 cells were preparedaccording to Bause-Niedrig, et al. {Bause-Niedrig, I., M. et al., Vet.Immunol. Immunopathol., 31:361-369 (1992)}. Proteins from these lysates(30 μg) and recombinant BDV proteins (250 ng) were subjected to 12%SDS-PAGE {Laemmli, U. K., et al., J. Mol. Biol., 80:575-581 (1973)} andthen transferred to nitrocellulose membranes (Schleicher & Schuell,Keene, N.H.) {Towbin, H., et al., Proc. Natl. Acad. Sci. USA,76:4350-4354 (1979)}. Membranes were incubated at room temperature firstwith WB-diluent (0.5% nonfat dry milk (Carnation Company, Los Angeles,Calif.) and 0.05% Tween-20 (Fisher Scientific, Raleigh, N.C.) in TBS(tris balanced saline, 50 mM Tris-HCl pH 7.5 and 150 mM NaCl)) for onehour, then overnight with various dilutions (1:10 to 1:2,000) of ratsera or monospecific rabbit sera in WB-diluent. Membranes were washed 3times in TBS, incubated for 2 hours with the appropriate secondaryantibody (horseradish peroxidase-conjugated goat anti-rat IgG and IgM orgoat anti-rabbit IgG, Sigma Chemical Co., St. Louis, Mo.) diluted 1:500in WB-diluent, washed 5 times in TBS and then incubated with hydrogenperoxide and 4-chloro-1-naphthol (Pierce Chemical Company, Rockford,Ill.) according to manufacturer's instructions. Methods for synthesisand analysis of radiolabeled BDV proteins and iminunoprecipitation havebeen described {Lipkin, W. I., et al., Proc. Natl. Acad. Sci., USA,87:4184-4188 (1990)}. Briefly, plasmid clones pBDV-gp18, pBDV-23 andpBDV-40 were linearized and used as template for in vitro transcriptionand translation of [³⁵ S] methionine-labeled proteins. Afterprecipitation with rat or rabbit sera and protein A-sepharose (SigmaChemical Co., St. Louis, Mo.), proteins were analyzed by SDS-PAGE andautoradiography.

ELISA

Ninety-six well, Immulon I microtiter plates with lids (DynatechLaboratories, Chantilly, Va.) were coated overnight at 37° C. with 10 ngof recombinant protein per well in 100 μl of borate buffer (100 mM boricacid, 50 mM sodium borate and 75 mM sodium chloride, pH 8.4). Plateswere washed three times with washing buffer (0.05% Tween-20 in PBS) andincubated for 1 hour at 37° C. with ELISA-diluent (0.5% bovine serumalbumin (BSA) fraction V (USB) in washing buffer). Two-fold serialdilutions of sera were prepared in ELISA-diluent; 100 μl of sera dilutedfrom 1:250 to 1:500,000 was then added to each well and incubated for 2hours at 37° C. Plates were washed three times with washing buffer.Next, 100 μl of horseradish peroxidase-conjugated goat anti-rat IgG andIgM (Sigma Chemical Co., St. Louis, Mo.) diluted 1:5,000 inELISA-diluent were added to each well and incubated for 1 hour at 37° C.After washing the plates five times, 100 μl of substrate solution wasadded to each well. Substrate solution consisted of 9.9 ml of 100 mMsodium acetate adjusted to pH 6.0 with 100 mM citric acid, 100 μl of 10mg of 3,3',5,5'-tetramethylbenzidine (Sigma Chemical Co., St. Louis,Mo.) per ml in dimethyl sulfoxide and 1.5 μl of 30% hydrogen peroxide(Fisher Scientific, Raleigh, N.C.). After incubation in the dark at roomtemperature for 30 minutes, the reaction was stopped by the addition of50 μl of 25% sulphuric acid (Sigma Chemical Co., St. Louis, Mo.) to eachwell. The absorbance at 450 nm was determined for each well using amicroplate reader (Molecular Devices, Thermo max, Menlo Park, Calif.).Negative control wells, without primary antisera, were used forcalibration. The ELISA titer for each serum was defined as the endpointdilution that yielded an optical density of 0.3.

RESULTS

The figures below present some of the results:

FIG. 11. Western blot analysis of native and recombinant proteins withmonospecific antisera to recombinant proteins and sera from infectedrats. Recombinant viral proteins and lysates from infected C6BDV ornoninfected C6BDV cells were size-fractionated and screened by Westernblot. A) Sera from infected and noninfected rats were used to detectnative or recombinant proteins. Lane 1, C6BDV lysate; lane 2, recp40;lane 3, recp23; lane 4, recp18; lane 5, C6 lysate; lane 6, recp40,recp23 and recp18. Lanes 1-4 were treated with serum from infected rat;lanes 5 and 6 were treated with serum from noninfected rat. B)Monospecific antisera were used to detect BDV-specific proteins. C6BDVlysates (lanes 1-3) and C6 lysates (lanes 4 and 5) were incubated with:lanes 1 and 4, serum from infected rat; lane 2, anti-p40 rabbit serum;lane 3, anti-p23 rabbit serum; and lane 5, pooled anti-p40 and anti-p23sera.

FIG. 12. ELISA of infected rat serum reacted with recp40. ELISA wasperformed with 10 ng/well recp40 or BSA as described in Materials andMethods. Circles, recp40 and serum from chronically infected rat;squares, recp40 and serum from noninfected rat; triangles, BSA and serumfrom chronically infected rat.

FIG. 13. Timecourse for appearance of antibodies to BDV-proteins. Serawere collected at different times post-infection and assayed by ELISAfor antibodies to (A) recp40; (B) recp23; and (C) recp18. Error barsrepresent standard error of the mean. Number of animals analyzed at eachtime point: <4 wks, 15; 5 wks, 6; 6 wks, 12; 8 wks, 4; 10 wks, 5; and 15wks, 9.

Production of Recombinant Viral Proteins and Monospecific Antisera toRecombinant Viral Proteins

Full length coding sequences for p40, p23 and gp18 were expressed inEscherischia coli and recombinant proteins were purified. The yield ofprotein in 100 ml of bacterial culture was: recp40, 1 mg; recp23, 500μg; and recp18, 50 μg. Recombinant proteins were analyzed by SDS-PAGE. Apredominant band of the expected molecular weight was observed for eachprotein and tested for antigenicity by WB using sera from BDV-infectedand noninfected rats (FIG. 11A). Recombinant proteins were detected bysera from BDV-infected rats but not by sera from noninfected rats.Recombinant proteins, recp40 and recp23 were used to produce antibodiesin rabbits. The production of antibodies was monitored by ELISA. Rabbitswere sacrificed when the ELISA titer reached 1:500,000 (week 16 ofimmunization). The specificity of the antisera was then tested by WBusing lysates from infected cells and recombinant proteins (FIG. 11B).Antisera were monospecific: rabbits immunized with recp40 producedantibodies that reacted only with p40 and recp40; rabbits immunized withrecp23 produced antibodies that reacted only with p23 and recp23. Atweek 16 of immunization, the antisera were also titered by IFT. Antiserato recp40 and recp23 had IFT titers of 1:50,000 and 1:100,000,respectively.

Specificity and Sensitivity Demonstrated in the BDV-ELISA Systems

In order to establish a sensitive and specific ELISA for all threerecombinant BDV proteins, the optimal antigen concentration wasdetermined by checkerboard titration of positive and negative seraversus various antigen concentrations. For each protein, theconcentration that resulted in the most linear response was 10 ng/well.The sensitivity of the ELISA system for each recombinant protein wasestablished using sera from infected rats known to be reactive by IFT,IP and WB. For each of the proteins, 100% of sera that had been found tobe positive by other methods were also positive by ELISA. Specificitywas tested using sera from 15 noninfected rats. ELISA for each proteinproved to be highly specific for detection of antibodies to BDVproteins: recp40-ELISA with noninfected rat sera showed 80% specificityat 1:500 dilution or 100% specificity at 1:2,000, recp23-ELISA showed93% specificity at 1:250 and 100% specificity at 1:1,000, recp18-ELISAshowed 100% specificity at 1:250. FIG. 12 shows a representative ELISAusing recp40 as target antigen. Various dilutions of sera fromchronically infected and noninfected rats were tested with 10 ng ofrecombinant protein or BSA per well in comparison with BSA. Nononspecific background reactivity was observed at serum dilutions of1:500 or higher (FIG. 12). Results were similar when recp23 and recp18were used as target antigen.

Analysis of Immunoreactivity to Viral Proteins bv IFT WB, IP and ELISAin Sera from Infected Rats

Adult rats infected intranasally with BDV did not display abnormalbehaviors prior to the fourth week post-infection (predisease, PD). Fourto six weeks post-infection, in the acute phase of disease (AD), animalshad hyperactivity, weight loss, disheveled fur, dystonic posture andhindlimb paresis. Eight to fifteen weeks post-infection, signs ofdisease stabilized: there was no additional weight loss, hyperactivitydiminished and paresis did not progress. This chronic phase of thedisease (CD) persisted for the life of the animals. Sera was collectedfrom adult-infected rats between 3 and 15 weeks after infection withBDV, and analyzed for the presence of antibodies to viral proteins usingfour different methods: IFT, WB, IP and ELISA (Table 3).

                                      TABLE 3                                     __________________________________________________________________________    Detection of BDV-specific antibodies in sera from infected rats by            different methods                                                                        WB          IP.sup.a Reciprocal ELISA titer.sup.b                                                                         Reciprocal             Serum      recp40                                                                            recp23                                                                            recp18                                                                            p40                                                                              p23                                                                              p18                                                                              recp40  recp23  recp18 IFT                    __________________________________________________________________________                                                           titer                  PD (3-4 wk pi.sup.c ; n = 15)                                                            -   -   -   -  -  -  2,388 ± 256                                                                        904 ± 181                                                                          163 ± 5.sup.d                                                                     <10                    AD (4-6 wk pi; n = 18)                                                                   +   +   -   +  +  -  3,217 ± 829                                                                        2,644 ± 20                                                                         279 ± 19                                                                          20-200                 CD (10-15 wk pi;                                                                         +   +   +   +  +  +  291,889 ± 56,590                                                                   76,527 ± 13,309                                                                    4,680 ± 1,467                                                                     10,000-20,000          n = 14)                                                                       __________________________________________________________________________     .sup.a In vitrotranslated proteins.                                           .sup.b Values are mean ± standard error of the mean titer.                 .sup.c pi, postinfection.                                                     .sup.d Nonspecific, Value below the level of specificity of the recp18        ELISA (1:250).                                                           

IFT allowed detection of antibodies to BDV in both AD rats and CD rats.In AD rats, the titer was between 1:20 and 1:200, whereas in CD rats,the titer was between 1:10,000 and 1:20,000. Sera from PD rats were notreactive by IFT. WB using lysates from infected cells or recombinantproteins, and IP using proteins translated in vitro yielded identicalresults: sera from CD animals were reactive with p40, p23 and gp18; serafrom AD rats detected only p40 and p23; sera from PD rats did not reactwith p40, p23 or gp18. ELISA detected antibodies reactive with p40, p23and gp18 in sera from all CD and AD rats (Table 3). In PD rats, ELISAonly detected antibodies reactive with p40 and p23; immunoreactivitywith gp18 was below specificity (Table 3).

The timecourse for the appearance of antibodies to BDV-proteins in serawas determined by ELISA. Sera collected at regular intervals fromadult-infected rats were tested in the recp40, recp23 and recp18 ELISAsystems. Titers of antibodies to all three proteins increased throughoutthe period of observation from weeks 4 to 15 post infection (FIG. 13).

DISCUSSION

Three recombinant BDV proteins, recp40, recp23 and recp18, wereexpressed and used as immunogens for production of monospecific sera inrabbits. Two of these antisera, directed against recp40 and recp23, arereported here; antisera to recp18 are described in Example 4 below.These three recombinant proteins were detected by sera from infectedrats (FIG. 11A) and by monoclonal antibodies to purified nativeproteins. Monospecific antisera to the recombinant proteins wereimmunogen-specific as determined by WB (FIG. 11B) and detected proteinsin infected cells by IFT.

ELISA systems were established, based on recombinant proteins, that haveseveral advantages over methods currently used for detection ofBDV-specific antibodies including IFT, WB and IP. Although IFT is widelyaccepted as a method for diagnosing BDV infection and titeringantibodies to the virus, it has two disdavantages. First, IFT does notdefine the viral protein(s) responsible for immunoreactivity. Second, asshown here, IFT titers are 10-100 fold less sensitive than ELISA fordetection of antibodies to p40 or p23. This relative insensitivityresulted in failure of IFT to show evidence of infection in PD rats(Table 3). WB and IP allowed detection of antibodies to individual viralproteins but were also less sensitive than ELISA. Sera from PD rats werenot reactive by either WB or IP.

For diagnostic purposes, the recp40-ELISA is the most sensitive methodfor detection of antibodies in infected animals. Antibodies to recp40were present prior to disease onset and had higher titers thanantibodies to recp23 or recp18. Although the recp23-ELISA was alsopositive in PD and AD rats, the recp18-ELISA was not. Because high titerantibodies to gp18 only appear in chronic disease, the recp18-ELISA maybe used to estimate the duration of infection. Low antibody titers torecp18 are not due to the lack of glycosylation on this recombinantprotein because similar ELISA titers were found with native gp18antigen. Failure to produce high titer antibody response to recp18 maybe due to lower levels of expression of this protein than p40 or p23.

Growing recognition that BDV has a broader species and geographic rangethan previously appreciated suggests the importance of designingsensitive, reliable assays for infection. The ELISA systems describedhere, provide inexpensive, rapid methods for BDV-serology. In contrastto IFT, WB and IP, which require at least 2 days for completion and arenot well suited to screening multiple samples, ELISA allows analysis ofhundreds of sera in several hours with only minimal equipment. Platescoated with these proteins have been stable in ELISA for up to one monthat room temperature and thus are practical for use in remotelaboratories. In addition to serving as a tool for clinical diagnosisand epidemiology of Borna disease infection, the BDV ELISA is a usefultool for studies in immunopathogenesis and virus biology. For example,applicants have mapped antigen binding sites on p40 and p23 by ELISAusing sera from infected animals and monoclonal antibodies to BDVproteins.

Dependent on the population studied and the methods used for analysis(WB, IP or IFT), the prevalence of antibodies reactive with BDV proteinsin patients with neuropsychiatric disorders has been estimated to bebetween 4% and 23% {Bode, L., In W. I. Lipkin and H. Koprowski (ed.),Borna Disease. Springer-Verlag, Heidelberg, in press (1995)}.Variability between laboratories could be due to differences inpopulations analyzed, antigen preparations or experimental technique.The BDV ELISA based on recombinant proteins provides a standardizedmethod for investigating human immunoreactivity to this neurotropicinfectious agent.

EXAMPLE 4 Neutralizing Antibodies in BDV Infected Animals

We examined the timecourse for the development of neutralizationactivity and the expression of antibodies to individual BDV viralproteins in sera of infected rats. The appearance of neutralizingactivity correlated with the development of immunoreactivity to gp18,but not p40 or p23. Monospecific and monoclonal antibodies to nativegp18 and recombinant non-glycosylated gp18 were also found to haveneutralizing activity and to immunoprecipitate viral particles orsubparticles. These findings suggest that gp18 is likely to be presenton the surface of the viral particles and to contain epitopes importantfor virus neutralization.

Antibodies to p40 and p23 (soluble antigens) are readily detected inboth sera and cerebrospinal fluid (CSF) of naturally and experimentallyinfected animals {Ludwig, H., et al., Progr. Med. Virol., 35:107-151(1988); Ludwig, H., et al., Arch. Virol., 55:209-223 (1977) and Ludwig,H., et al., Med. Microbiol. Immunol., 163:215-226 (1977)}. Antibodies togp18, a membrane-associated glycoprotein (previously described as 14.5kDa), have been reported less frequently {Ludwig, H., et al., Progr.Med. Virol., 35:107-151 (1988) and Rubin, S. A., et al., J. Virol.,67:548-52 (1993)}. Although neutralization activity has been found insera of animals infected with BDV {Danner, K., et al., Zbl. Vet.-Med.[B], 25:345-355 (1978); Hirano, N., et al., J. Gen. Virol., 64:1521-1530(1983); Ludwig, H., et al., Progr. Med. Virol., 35:107-151 (1988) andLudwig, H., et al., Arch. Virol. [Suppl] 7:111-133 (1993)}, theantibodies responsible for neutralization activity have not beeninvestigated. An enzyme-linked immunosorbent assay (ELISA) based onrecombinant BDV proteins has been established in Example 3 above, thatprovides a sensitive method for detection of antibodies to gp18. We findthat the appearance of neutralizing antibodies in infected ratscorrelates with immunological reactivity to gp18. Furthermore,monospecific and monoclonal antibodies (MAbs) directed against gp18neutralize BDV infectivity and immunoprecipitate viral particles orsubparticles.

MATERIALS AND METHODS

BDV infected animals: Sixty-thousand focus forming units (ffu) of BDVstrain He/80-1 {Carbone, K. M., et al., J. Virol., 61:3431-3440 (1987);Herzog, S., et al., Med. Microbiol. Immunol., 168:158-8 (1980) andSchneider, P. A., et al., J. Virol., 68:63-68 (1994)} were used tointranasally (i.n.) infect each of seventy 6-week old Lewis rats. Ratswere observed at three days intervals for weight loss, ruffled fur orpostural abnormalities consistent with acute disease. Sera werecollected at time of sacrifice. Under metofane anesthesia, rats wereperfused with buffered 4% paraformaldehyde; brains were fixed overnightin perfusate at 4° C. Twenty-micron sagittal sections were collectedonto gelatin coated slides and stained with hematoxylin and eosin.Inflammation was scored using the scale of Stitz, Sobbe and Bilzer{Stitz, L., et al., J. Virol., 66:3316-23 (1992)}.

Virus Titration and Neutralization Assay

Viral infectivity in 20% brain homogenates was determined using themethod of Pauli et al. {Pauli, G., et al., Zbl. Vet.-Med. [B] 31:552-557(1984)}. Virus neutralization was performed using a modification ofDanner et al. {Danner, K., et al., Zbl. Vet.-Med. [B], 25:345-355(1978)}. Briefly, 50 ffu of BDV were incubated with serial dilutions ofantibodies or sera for one hour at 37° C., added to rabbit fetal glialcells and incubated for 5 days. Sera was heat inactivated at 56° C. for30 minutes. In selected assays, mouse complement (1:50) (Sigma ChemicalCo., St. Louis, Mo.) was added to the virus concurrent with the additionof MAbs to determine the effects of complement on neutralizationactivity. The dilution of serum or antibody required to reduce thenumber of ffu by 50% was defined as the neutralization titer (NT₅₀). Ascontrols for each neutralization assay, rabbit fetal cells were exposedto medium without virus, treated with virus in medium alone (noantibodies), or treated with virus incubated with sera from normal rats.Pilot studies showed that approximately 8% of normal rat sera interferedwith BDV infectivity at dilutions up to 1:16. Therefore, sera wereconsidered to be neutralizing only if the NT₅₀ exceeded 1:32.Supernatant from nonproducing myeloma cell lines as well as monoclonalantibodies directed against BDV-p23 (24/36F1) and BDV-p40 (38/17C1){Thiedemann, N., et al., J. Gen. Virol., 73:1057-1064 (1992)} were foundto neutralize infectivity at dilutions of 1:2. Thus, monoclonalantibodies were considered to be neutralizing only if the NT50 exceeded1:4.

Preparation of Proteins (recp40, recp23, recp18 and gp18)

Plasmids encoding p40 {pBDV-40 disclosed in McClure, M. A., et al., J.Virol., 66:6572-6577 (1992)}, p23 {pBDV-23 disclosed in Thibault, K. J.,M.S. thesis; University of California, Irvine (1992)} and gp18{pBDV-gp18 disclosed in Kliche, S. et al., J. Virol., 68:6918-6923 andExample 2 above} were subcloned (see Example 3 above) into theprokaryotic expression vector petl5b (Novagen, Madison, Wis.).Recombinant proteins (recp40, recp23 and recp18) were expressed inEscherichia coli and purified according to manufacturer's protocol(Novagen, Madison, Wis.). Purity and antigenicity were assessed bySDS-PAGE and Western blot analysis using sera from infected rats.Native, glycosylated gp18 was prepared from infected rat brain asdescribed previously {Schadler, R., et al., J. Gen. Virol., 66:2479-2484(1985)}.

Enzyme-Linked Immunosorbent Assay (ELISA)

ELISA was performed as described in Example 3 above. Briefly, platescoated with recombinant protein were incubated with serially dilutedsera or MAbs. Bound horseradish peroxidase (HRPO)-coupled secondaryantibody (goat anti-mouse F'ab-HRPO, goat anti-rat IgG and IgM HRPO;Sigma Chemical Co.) was quantified on a microplate reader (Thermo max,Molecular Devices, Menlo Park, Calif.) using the chromagen 3,3'-5,5'Tetramethylbenzidine (Sigma Chemical Co.). The ELISA endpoint titer wasdefined as the serum or antibody dilution that generated an opticaldensity of 0.3.

Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE),Western Blot and Immunoprecipitation (IP)

Recombinant or native BDV proteins were subjected to SDS-PAGE {Laemmli,U. K., et al., J. Mol. Biol., 80:575-581 (1973)} and transferred tonitrocellulose (Schleicher & Schuell, Inc., Keene, N.H.) or Immobilon-Nmembranes (Millipore Corp., Bedford, Md.) {Towbin, H., et al., Proc.Natl. Acad. Sci. USA, 76:4350-4354 (1979)}. Membranes were blocked andincubated with primary antibody as described in Example 3 above. Afterincubation with secondary antibody (goat antimouse IgG-alkalinephosphatase [AP], goat anti-rat IgG-AP or goat anti-rat IgG andIgM-HRPO, Sigma Chemical Co., St. Louis, Mo.), immune complexes werevisualized using Western Blue (Promega, Madison, Wis.) for AP orchemiluminescence (ECL kit, Amersham, Arlington Heights, Ill.) for HRPOaccording to manufacturer's instructions. gp18 or recp18 wereprecipitated using sera from infected rats, monospecific antibodies orMAbs and Protein A-Sepharose (Pharmacia Biotech Inc., Piscataway, N.J.)as described by Persson, H., et al. {Persson, H., et al. Science,225:687-693 (1984)} then assayed by Western blot.

Monoclonal Antibodies

MAbs to gp18 were generated according to Thiedemann et al. {Thiedemann,N., et al., J. Gen. Virol., 73:1057-1064 (1992)}. Briefly, Balb/c micewere immunized intraperitoneally (i.p.) with 5 μg of gp18 in completeFreund's adjuvant. Three and 6 weeks after the initial immunization,mice were boosted i.p. with 5 μg of gp18 in incomplete Freund'sadjuvant. Four days before fusion of spleen cells with the mouse myelomacells X63-Ag8.653 {Kearney, J. F., et al., J. Immunol., 123:1548-1550(1979)}, mice were boosted intravenously with 15 μg of gp18. Allhybridomas were initially screened for reactivity to gp18 by ELISA.Tissue culture supernatants from positive hybridomas were concentratedby ammonium sulfate precipitation {Jonak, Z. L., p. 405-406, In R. H.Kennett, T. J. McKean, and K. B. Becktol (ed.), "Monoclonal antibodies,Hybridomas: A new dimension in biological analyses", Plenum Press, NewYork (1982)} and tested by Western blot and IP for reactivity with gp18and recp18. The immunoglobulin isotype was determined using anagglutination isotyping kit (Serotec, Oxford, England) according tomanufacturer's instructions. Monoclonal antibody, 24/36F1 directedagainst BDV-p23 {Thiedemann, N., et al., J. Gen. Virol., 73:1057-1064(1992)}, was used as a negative control in Western blot and IPexperiments.

Generation of Polyclonal Sera Against Recp18 Protein

To produce antibodies against recp18, two 2-month old Lewis rats wereinjected subcutaneously (s.c.) with 25 μg of protein in Freund'scomplete adjuvant and boosted 3 weeks later with 25 μg of protein s.c.in Freund's incomplete adjuvant. After 6 weeks, animals received i.p.injections of 25 μg protein in phosphate buffered saline (PBS) with 20μg lipopolysaccharide (S. typhimurium, Difco, Detroit, Mich.) attwo-week intervals for a total of three injections. Serum was collectedevery two weeks during weeks 7 through 14 for analysis by ELISA andWestern blot and for determination of neutralization titer. Mouseantibodies to native gp18 have been described in Example 2 above.

Affinity Adsorption of BDV-Specific Serum-Antibodies

Antibodies that bound to recp23 and recp40 were sequentially removedfrom serum of an infected rat according to Crabb et. al. {Crabb, B. S.,et al., Virology, 190:143-154 (1992)}. Serum (D2) from an adult-infectedLewis rat (15 weeks post intranasal infection), was diluted 1:10 in TBS(tris balanced saline, 50 mM Tris pH 7.4 and 100 mM NACl) and incubatedovernight at 4° C. with membrane-bound recp23. The anti-recp23antibody-depleted serum (D2 Δ∝recp23) was removed, the membrane waswashed with TBS and adsorbed anti-recp23 antibodies were eluted (recp23eluant) by incubation with 1 ml of 0.1 M glycine, 0.15 M NaCl pH 2.7 for3 minutes. The pH of the eluant was adjusted by addition of 300 μl of 10mM Tris HCl pH 7.5. The anti-recp23 antibody-depleted serum was thenincubated with membrane-bound recp40 (D2 Δ∝recp23, Δ∝recp40) andpurified as before (recp40 eluant). Antibody depletion from serum andantibody elution from membrane-bound proteins was monitored by Westernblot and ELISA. At each step during the purification, antibody-depletedsera and eluted antibodies were analyzed for neutralizing activity.Antibodies to gp18 or recp18 were also adsorbed (D2 Δ∝gp18, D2 Δ∝recp18)and eluted (gp18 eluant, recp18 eluant) by this method. These adsorptionand elution experiments were repeated using serum (B3) from anadditional adult-infected rat (15 week post intranasal infection).

IP of BDV Particles or Sub Particles and Analysis by ReverseTranscription Polymerase Chain Reaction (PCR)

Forty-thousand ffu of BDV in a volume of 200 μl were treated with 50μg/ml of DNase I and RNase A (Boehringer Mannheim Corp., Indianapolis,Ind.) for 30 minutes at 37° C. then incubated for 2 hours at roomtemperature with 100 μl of one of the following: (1) serum from acutelyor chronically infected rats at 1:10 dilution in PBS; (2) purifiedserum-antibodies at 1:10 dilution; (3) mouse anti-gp18 sera or ratanti-recp18 sera at 1:20 dilution; or (4) monoclonal antibodies againstgp18 at 1:5 dilution. Next, 100 μl of 1 mg/ml Protein A-Sepharose(Pharmacia, Puscataway, N.J.) in PBS was added, and the mixture wasincubated overnight at 4° C. The Protein A-Sepharose-antibody-viruscomplex was washed three times in PBS then resuspended in 100 μl water.Total RNA was extracted {Chomczynski, P., et al., Anal. Biochem., 162:156-159 (1987)} and used for RT-PCR amplification of a 693 nucleotideregion of the viral genome (nucleotide 753 to 1446) according toSchneider et al. (primer 7 and primer 9) {Schneider, P. A., et al., J.Virol., 68:63-68 (1994)}. PCR products were analyzed by agarose gelelectrophoresis. PCR products were cloned and sequenced to confirm thatthey represented the predicted region of the genomic RNA {Schneider, P.A., et al., J. Virol., 68:63-68 (1994)}. Negative controls for RT-PCRincluded the omission of virus from immunoprecipitation reactions andthe use of genomic sense primers during first strand cDNA synthesis.

RESULTS

The following figures present part of the results:

FIG. 14. Timecourse for the appearance of antibodies to BDV proteins insera from individual rats after i.n. infection. (A) Neutralizationactivity in sera from BDV-infected rats at three time points (5, 10 and15 weeks post-infection). Each serum is represented by a circle. Barsindicate mean neutralization titer for each group (5, 10 or 15 weekspost-infection). Asterisk represents sera with neutralization titer lessthan or equal to 1:16. (B) Plot of mean recp18 ELISA titers (opencolumns) with neutralization titers (hatched columns) at three timepoints (5, 10 and 15 weeks post-infection). Sera analyzed were the sameas those in panel A. Mean values for neutralization activity weredetermined as described in FIG. 14A. Arrows indicate threshold forsignificance in neutralization assay (1:32) and recp18 ELISA (1:250).These values were selected because normal rat sera reacted in theneutralization assay and recp18 ELISA at titers of 1:16 and 1:125,respectively. (C) Timecourse for the appearance of antibodies to recp40,recp23, and gp18 by Western blot analysis. Proteins weresize-fractionated by SDS-PAGE and transferred to nitrocellulosemembranes. Membranes were incubated first with sera and then withhorseradish peroxidase-coupled goat anti-rat IgG. Bound secondaryantibody was detected by chemiluminescence. Results shown are from serumof one representative animal at several different timepoints post BDVinfection (p.i.).

FIG. 15. Monoclonal antibody (MAb) detection of gp18. A)Immunoprecipitation of gp18 with MAbs. gp18 was first incubated withMAbs or sera from infected or noninfected rats, then precipitated withProtein-A Sepharose, size-fractionated by 12% SDS-PAGE and transferredto Immobilon-N membranes. Precipitated gp18 was visualized with ratanti-recp18 sera, goat anti-rat IgG-AP, and Western Blue. Lanes 1, serumfrom infected rat (15 week p.i.); 2, serum from noninfected rat; 3, MAb14/29A5; 4, MAb 14/26B9; 5, MAb 14/8E1; 6, MAb 14/13E10; 7, MAb 14/18H7;8, MAb 24/36F1 (MAb directed against the BDV 23 kDa protein, negativecontrol); 9, no antibody. Arrow indicates gp18; H and L represent heavyand light chains of immunoglobulin, respectively. B) MAbs were analyzedfor binding to native gp18 in Western blot. gp18 was separated on 12%SDS-PAGE and transferred to an Immobilon-N membrane. Strips wereincubated with MAbs or sera from infected or noninfected rats. Boundantibodies were detected with alkaline phosphatase conjugated goatanti-rat IgG or goat anti-mouse Fab-specific and Western Blue substrate.Lanes: 1, serum from infected rat (15 week p.i., D2); 2, serum fromnoninfected rat; 3, MAb 14/29A5; 4, MAb 14/26B9; 5, MAb 14/8E1; 6, MAb14/13E10; 7, MAb 14/18H7; and 8, MAb 24/36F1 (MAb directed against theBDV 23 kDa protein, negative control). Molecular weight markers (10³ Da)are shown at the right.

FIG. 16. Neutralization profile of sera and MAbs. BDV (50 ffu) waspreincubated with serial dilutions of serum or MAb and then added to tenthousand rabbit fetal glial cells. After four days of incubation, theinfected cells were visualized as described in Pauli et al. {Pauli, G.,et al., Zbl. Vet.-Med. [B] 31:552-557 (1984)}. The number of infectedcell-foci per well was counted. (A) Serum from noninfected rat. (B)serum from infected rat (15 week p.i., D2). (C) MAb 14/13E10. (D) MAb14/29A5.

FIG. 17. Precipitation of BDV using sera from infected rats,monospecific rat antisera to recp18 and monoclonal antibodies (MAbs) togp18. Virus was treated with nucleases to eliminate nucleic acid notcontained within virions then immunoprecipitated with sera or MAbs andProtein A-Sepharose. RNA was extracted and subjected to RT-PCR toamplify a 693 nucleotide viral genomic sequence. PCR-products werevisualized in an ethidium bromide-stained 1% agarose gel. (A)Precipitation of BDV with sera from infected rats. Lanes: 1, serum frominfected rat, 15 week p.i.; 2, serum from infected rat, 5 week p.i.; 3,serum from infected rat, 15 week p.i., no BDV; 4, serum from infectedrat, 15 week p.i., genome sense primer used for first strand cDNAsynthesis. (B) Precipitation of BDV by monospecific antisera to recp18and MAbs to gp18. Lanes: 1, monospecific rat antisera to recp18; 2, MAb14/13E10; 3, MAb 14/29A5. DNA markers (basepairs) are shown at theright.

Timecourse of Disease and Appearance of Antibodies to BDV in InfectedRates

Rats developed Borna disease (BD) within 5 weeks post infection. Theacute phase of the disease, 4-8 weeks post infection, was associatedwith marked weight loss, disheveled fur, dystonic posture, hind limbparesis and paralysis, mortality of 35%, and prominent inflammatory cellinfiltrates in the brain. In the chronic phase of disease, 10-15 weekspost-infection, signs of disease stabilized and inflammation receded.Virus titers in the brains of animals acutely (5 weeks p.i.) andchronically infected (15 weeks p.i.) were 2.4±0.4×10⁵ ffu/ml and4.4±0.2×10⁴ ffu/ml, respectively.

Sera were monitored for virus neutralization activity (FIGS. 14A, B andC) and the presence of antibodies reactive with recp40, recp23, recp18or native gp18 in Western blot (FIG. 14C) and ELISA. Neutralizationactivity was first detected in sera (28% of the animals) at 5 weeks p.i.By week 15 p.i., all sera had neutralization activity with a mean titerof 1:977±246. Antibodies to recp18 were first detected by ELISA at week5 p.i. and showed a marked increase in titer by 15 weeks p.i.(1:4,610±1,463) (FIG. 14B). In contrast, antibodies reactive with recp40and recp23 were detected by ELISA within 4 weeks of infection, reached atiter greater than 1:20,000 by 8 weeks p.i. and remained elevatedthrough 15 weeks p.i. (see Example 3 above). Antibodies reactive withrecp40 and recp23 were detected by Western blot between weeks four andfive p.i., whereas antibodies to gp18 were detectable only after week 10p.i. (FIG. 14C).

Affinity Adsorption of Neutralizing Sera

To determine whether the presence of antibodies to gp18 correlate withneutralization activity, two rat sera (D2 and B3, 15 weeks p.i.), weretested in the neutralization assay after successive depletions ofantibodies to individual BDV-proteins. Antibodies to BDV-specificproteins were removed from D2 rat serum by adsorption withmembrane-bound protein. The efficiency of antibody depletion from serumwas monitored by Western blot and ELISA. Prior to adsorption, the titersto recp40 and recp23 were each greater than 1:20,000. Followingadsorption with recp23, the titer to recp23 decreased to 1:200. Afteradsorption with recp40, the titer to recp40 decreased to 1:150. Elutedantibodies were reactive by ELISA with the proteins used for adsorption:recp23 eluant titer, 1:5,000; recp40 eluant titer, 1:15,000. Serumantibodies remaining after adsorption, and eluted antibodies, were thentested for neutralizing activity. The neutralization titer of the D2serum (NT₅₀ 1:1,000-1,500) did not change after adsorption with recp23and recp40 antigens (D2 Δ∝recp23, Δ∝recp40) (Table 4). Antibodies elutedfrom proteins recp40 (recp40 eluant) and recp23 (recp23 eluant) had noneutralization activity (Table 4). In contrast, the NT₅₀ of the D2 serumdecreased from 1:1,000-1,500 to 1:600-700 after adsorption with recp18(D2 Δ∝recp18) and to 1:160-200 after adsorption with gp18 (D2 Δ∝gp18)(Table 5). The neutralization titers of antibodies eluted from recp18(recp18 eluant) and gp18 (gp18 eluant) were 1:60-100 and 1:240-400,respectively (Table 4). Similar results were obtained with serum fromrat B3.

                  TABLE 4                                                         ______________________________________                                        Characterization of serum antibodies                                                                             Reciprocal                                               Reciprocal           rp18 ELISA                                 Serum         NT        IP--RT--PCR.sup.a                                                                        titer                                      ______________________________________                                        Chronic (15 wk p.i. [D2])                                                                   1,000-1,500                                                                             +          4,300-5,000                                D2 Δαrecp23, Δαrecp40.sup.b                                         1,000-1,500                                                                             +          4,000-5,000                                D2 Δαrecp18.sup.b                                                               600-700   +          NS.sup.c                                   D2 Δαgp18.sup.b                                                                 160-200   +          800-840                                    recp18 eluant.sup.d                                                                          60-100   +          2,400-3,000                                gp18 eluant.sup.d                                                                           240-400   +          1,200-2,000                                recp23 eluant.sup.d                                                                         NS        -          NS                                         recp40 eluant.sup.d                                                                         NS        -          NS                                         Ratαrecp18                                                                            320-480   +          >5,000                                     Mouseαgp18                                                                            160-320   +          >5,000                                     ______________________________________                                         .sup.a IP of BDV and detection of genomic RNA by RT--PCR.                     .sup.b Chronic rat sera (D2) adsorbed with recombinant (recp23, recp40,       recp18) or native (gp18) protein.                                             .sup.c NS, not significant. The NT was considered to be not significant       below 1:32; the recp18 ELISA titer was considered to be not significant       below 1:250.                                                                  .sup.d Antibodies in chronic rat sera (D2) eluted from recombinant or         native protein.                                                          

                                      TABLE 5                                     __________________________________________________________________________    Characterization of anti-gp18 MAbs                                                           Characterization by:                                                    Reciprocal                                                                          Western blot                                                                        IP    Reciprocal rp18                                    MAb  Ig class                                                                          NT    gp18                                                                             rp18                                                                             gp18                                                                             rp18                                                                             ELISA titer                                                                           IP--RT--PCR*                               __________________________________________________________________________    14/29A5                                                                            IgG2b                                                                             >400-1,000                                                                          +  +  +  +    500-1,000                                                                           -                                          14/26B9                                                                            IgM 8-16  -  -  +  +  4-8     +                                          14/8E1                                                                             IgM 50    -  -  +  +  200-300 +                                          14/13E10                                                                           IgM 50-100                                                                              -  -  +  +  16-64   +                                          14/18H7                                                                            IgG3                                                                              100-200                                                                             -  -  +  +  128-256 +                                          __________________________________________________________________________     *IP of BDV and detection of genomic RNA by RT--PCR.                      

Monospecific Antibodies to recp18 and gp18

Sera from rats and mice immunized with recp18 and gp18, respectively,were tested for neutralization activity. Neutralizing antibodies in bothrats and mice were detected at 12 weeks post-immunization. Sixteen-weeksafter immunization with recp18, 2 rats had neutralization titers between1:320 and 1:480 (Table 4); sera from 2 mice immunized with gp18 hadneutralization titers between 1:160 and 1:320 (Table 4).

Monoclonal Antibodies to gp18

MAbs were generated against gp18. Five positive clones were identifiedby ELISA using gp18 as antigen. The MAbs represented three differentimmunoglobulin isotypes, yet all contained the kappa light chain (Table5). Although each of the monoclonal antibodies immunoprecipitated gp18(Table 5, FIG. 5A) and recp18 (Table 5), only MAb, 14/29A5 reacted byWestern blot (Table 5, FIG. 15B).

Concentrated supernatants from all five MAbs neutralized BDV infectivity(Table 5). Similar to sera from chronically-infected rats (FIG. 16B),the neutralization titer of four MAbs was greatest at highest antibodyconcentration (FIG. 16C). In contrast, one MAb, 14/29A5, neutralized BDVonly when used at a dilution of 1:400-1:1,000 (FIG. 16D). Neutralizingsera from chronically-infected rats, mice immunized with gp18 or ratsimmunized with recp18 (Table 4) had the capacity to inhibit 100% of BDVinfectivity (FIG. 16B). In contrast, MAbs to gp18, used individually orin concert, inhibited a maximum of 68% of BDV infectivity (FIGS. 16C andD). Supernatants of two MABS, 14/18H7, NT₅₀ 1:16 and 14/13ElO, NT₅₀1:32, showed cooperativity in neutralization assays; pooling of theseMAbs resulted in a higher neutralization titer (NT₅₀ 1:100-150). Todetermine the extent to which neutralization was complement-dependent,neutralization activity of MAbs was tested with addition of eitheractive or heat-inactivated mouse complement. No increase inneutralization titer was detected with addition of mouse complement.Serum from noninfected (normal) rats was not neutralizing at dilutionsgreater than 1:16 (FIG. 16A).

Immunoprecipitation of BDV with Neutralizing Antibodies

BDV stock was treated with nucleases to eliminate free nucleic acidsthen incubated with sera or MAbs and Protein A-Sepharose. RNA wasextracted from immunoprecipitated viral particles or subparticles andsubjected to RT-PCR for amplification of viral genomic RNA. Neutralizingrat sera (FIG. 17A), monospecific sera to recp18 (FIG. 17B) or gp18, andD2 serum antibodies eluted from recp18 or gp18 precipitated BDVparticles. Removal of antibodies to recp23, recp40, recp18 or gp18 didnot affect the capacity of neutralizing sera to precipitate viralparticles. Four MAbs also precipitated BDV (FIG. 17B and Table 5). OneMAb, 14/29A5, did not precipitate viral particles at any dilution (1:5,1:100, 1:200 or 1:500). Sera from noninfected or two acutely infectedrats (5 weeks p.i.) (FIG. 17A) did not precipitate BDV. Experiments withsera and monoclonal antibodies are summarized in Tables 4 and 5.Negative controls for RT-PCR included the omission of virus fromimmunoprecipitation (FIG. 17A) and the use of genomic sense primers forfirst strand cDNA synthesis (FIG. 17A).

DISCUSSION

The presence (or absence) of neutralizing antibodies in BDV-infectedanimals has been controversial. Some reports have not shown evidence forneutralizing antibodies {Carbone, K. M., et al., J. Virol., 61:3431-3440(1987); Herzog, S., et al., J. Gen. Virol., 66:503-8 (1985) and Narayan,O., et al., J. Inf. Dis., 148:305-315 (1983)}, however, this may reflectdifferent timepoints for collection of sera or variation in the assaysystem for neutralization. Although there are reports of neutralizingantibodies in serum and CSF of both naturally and experimentallyinfected animals {Danner, K., et al., Zbl. Vet.-Med. [B], 25:345-355(1978); Hirano, N., et al., J. Gen. Virol., 64:1521-1530 (1983); Ludwig,H., et al., Progr. Med. Virol., 35:107-151 (1988) and Ludwig, H., etal., Arch. Virol. [Suppl] 7:111-133 (1993)}, neither the timecourse fordevelopment of neutralizing antibodies nor their target antigens havebeen characterized. Here, we show that the neutralizing activity ofBDV-rat sera increases dramatically from the acute (5 weeks p.i.) to thechronic (15 weeks p.i.) phase of disease and provide evidence toindicate that neutralization activity is due, at least in part, toantibodies that react with a BDV glycoprotein, gp18. The timecourse forthe appearance of neutralizing antibodies seems to correlate withimmunoreactivity to gp18. Furthermore, removal of antibodies to gp18 orrecp18 dramatically decreased the neutralization titer of BDV-rat sera.In contrast, subtraction of antibodies to two other viral proteins, p40and p23, had no effect.

Neutralization activity was detected with monospecific antiserum againstboth gp18 and recp18 as well as with monoclonal antibodies against gp18.These MAbs represent three different isotypes, IgM, IgG2b and IgG3,indicating that multiple isotypes are capable of virus neutralization.Addition of complement did not enhance neutralization activity of theMAbs, suggesting that the mechanism for neutralization was neithercomplement-mediated inactivation of virus nor steric hindrance by acomplement-MAb-virus complex.

It is likely that at least three different antibody binding sites ongp18 were involved in neutralization. Four MAbs, whichimmunoprecipitated both gp18 and recp18 but did not detect protein inWestern blots, presumably bound to discontinuous epitopes. Theobservation that use of MAbs 14/13E10 and 14/18H7 in combination,resulted in greater neutralization activity than use of either MAbalone, suggests that these MAbs recognized either differentdiscontinuous epitopes or different binding sites on a singlediscontinuous epitope. One MAb, 14/29A5, detected protein in Westernblots as well as immunoprecipitation assays indicating that it bound toa continuous epitope. Unlike the other MAbs, 14/29A5 neutralizedinfectivity only after dilution (FIG. 16D), a profile consistent withneutralization by virus aggregation as reported in other viral systems{Dimmock, N. J., A. Capron, et al. (ed.), "Current Topics inMicrobiology and Immunology", Springer-Verlag, Berlin (1993) and Outlaw,M. C., et al., J. Gen. Virol., 71:69-76 (1990)}. Although all of thegp18 MAbs detected recp18 (nonglycosylated protein), it is possible thatthere are additional epitopes for neutralization which include thecarbohydrate portion of gp18.

Sera from chronically-infected rats had greater neutralization activitythan monospecific sera or monoclonal antibodies directed against gp18.Higher neutralization activity in sera from infected animals couldreflect factors that influence epitope presentation such as interactionsbetween gp18 and other proteins or the virion envelope. Alternatively,gp18 may not be the only BDV protein that elicits neutralizingantibodies. Sera from chronically-infected animals retained partialneutralizing activity and the capacity to precipitate virus afteradsorption with gp18. Although this may be due to incomplete subtractionof antibodies to gp18 (Table 4) neutralizing antibodies may be directedagainst other viral proteins as well. For example, an additionalcandidate for a virion surface protein that may elicit neutralizingantibodies is p57. This putative protein contains multiple potentialN-glycosylation sites and, as the product of the fourth ORF on the BDVgenome, is in the gene position generally occupied by glycoproteins inMononegavirales {Briese, T., et al., Proc. Natl. Acad. Sci. USA:91:4362-4366 (1994)}. It is contemplated that passive administration ofneutralizing antibodies or immunization with gp18 and other virionsurface proteins can alter BDV pathogenesis.

EXAMPLE 5 Fragments of Borna Disease Virus Proteins Immunoactive WithSera From Human Schizophrenics and BDV Infected Animals

The etiology of schizophrenia, a debilitating disease that affectsapproximately 1% of the world's population, is unknown. Higherprevalence in some geographic areas, seasonal variation in births ofsubjects who develop disease, increased risk of schizophrenia insubjects exposed to influenza virus during the second trimester in uteroand discordance for disease in monozygotic twins suggest the possibilityof an infectious basis {Kirch, D. G., Schizophrenia Bulletin, 19:355-370(1993)}. Borna disease virus has been implicated in human affectivedisorders by studies reporting that patients have serum antibodies toBDV {Rott, R., et al., Science, 228:755-756 (1985)} and the presence ofviral proteins and nucleic acids in peripheral blood mononuclear cells{Bode, L., et al., Nature Medicine, 1:232-236 (1995)}. Thecatecholamine-related stereotypic behaviors observed in BDV-infectedrats and catecholamine system dysfunction present in schizophreniaprompted Western blot (WB) studies of sera from schizophrenics forantibodies to BDV obtained from animals infected with the virus{Waltrip, R. W., II, et al., Psychiatry Res., 56:33-44 (1995)}. Thepresent invention discloses an ELISA test for schizophrenia and BDVinfection; fragments and peptides derived from p23 and gp18 which areimmunoreactive with sera from schizophrenics and animals infected withBDV and/or immunized with p23 and gp18. The test is specific, sensitive,fast, easy, and economical. For example, indirect immunofluorescenceassays (IFT) used in the current art does not define the viralprotein(s) responsible for immunoreactvity. IFT, immunoprecipitation(IP), and WB are also less sensitive than the ELISA of the presentinvention and require at least 2 days for completion and are unsuitablefor screening of multiple samples. In contrast, the present ELISAprovides inexpensive, rapid tests which allow analysis of hundreds ofserum samples, e.g. in several hours, with minimal equipment. Theadvantages of ELISA over the prior art diagnostic methods are describedin further detail in Briese, T., et al., J. Clin. Microbiol., 33:348-351(1995). Besides the detection of schizophrenia and BDV infectionsdisclosed in this Example, the ELISA method disclosed herein isgenerally applicable for studies and detection of neurologic andneuropsychiatric diseases and BDV infections in men and animals.

I. Immunoreactivity of p40, p23 and gp18, with Sera from Schizophrenics

Sera from 30 human schizophrenic patients were examined by ELISA forimmunoreactivity with recombinant BDV proteins N (recp40), P (recp23)and M (recp18) {The recombinant proteins were produced and the ELISAwere performed as described in Examples 3 and 4, above, which were alsodescribed Briese, T., et al., J. Clin. Microbiol., 33:348-351 (1995)}.Controls were sera from 30 age and sex matched normal subjects and 30patients with multiple sclerosis (MS), an autoimmune central nervoussystem (CNS) disease of unknown etiology. Although some sera detected Nor P but not M, all sera immunoreactive with M also detected N and P.Twenty-seven percent of schizophrenic subjects (7) had serum antibodiesto M, N and P versus 3% of normal subjects (1) or 0% of MS patients(p<0.0001) (Table 6). Immunoreactivity of sera with all 3 proteins wasconfirmed by WB using extracts from BDV-infected C6 cells supplementedwith gp18; IP of the recombinant BDV proteins and IFT using infectedrabbit fetal glial cells (the WB, IFT, and IP were conducted using themethods known in the art, as described in Briese, T., et al., J. Clin.Microbiol., 33:348-351 (1995)}). Sera not reactive with 1 or more of the3 proteins in ELISA were also negative in the other assays.

                  TABLE 6                                                         ______________________________________                                        Percentage of subjects with schizophrenia (SZ), multiple sclerosis (MS)       or                                                                            no neuropsychiatric disease (NND) immunoreactive in ELISA with                N, P and M proteins of Borna disease virus                                    N*        P†                                                                            M‡                                                                        N/P†                                                                         N/M‡                                                                     M/P†                                                                         N/P/M‡                   ______________________________________                                        SZ     32     47     27   33    27    27    27                                (n = 30)                                                                      MS     0      0      0    0     0     0     0                                 (n = 30)                                                                      NND    7      7      3    7     3     3     3                                 (n = 30)                                                                      ______________________________________                                         N = p40, P = p23, M = gp18                                                    *p < 0.01; †p < 0.001; ‡p < 0.0001                          N/P = N and P                                                                 N/M = N and M                                                                 M/P = M and P                                                                 N/P/M = N, P and M                                                       

II. Selection and Immunoreactivity of Truncated Fragments of p23 andgp18

The immunologic determinants on p23 and gp18 were determined usingtruncated fragments of these proteins. The fragments used are shown inFIGS. 20B and 21B. In FIG. 20B, the fragments are designated S1 to S4and NS, respectively. In FIG. 21B, the fragments are derived from theunglycosylated version of recp18, and are denoted M1 to M4 and MS. Thefragments are shown from the amino terminus (left) to the carboxylterminus (right) of the proteins. The numbers below each fragmentindicate the locations of the amino acids on the full length p23 orgp18, respectively. For example, p23 has a total of 201 amino acids,thus, as shown in FIG. 20B, S1 represents the full length p23 because itspans from amino acid at position 1 (denoted 1aa in the figure) to theamino acid at position 201 (denoted 201aa) of p23. S2 is a proteinrepresenting a fragment of p23, spanning from amino acid at position 37to position 201 of p23. Similarly, in FIG. 21B, MS is a proteinrepresenting a fragment of the unglycosylated gp18, spanning from aminoacid at position 1 to position 70 of the unglycosylated gp18. Thesefragments were recombinantly produced by selecting the appropriate PCRprimers for the fragments based on the cDNA of p23 and gp18 disclosed inthis patent application and by cloning the respective cDNA fragmentsinto the prokaryotic expression vector pET15b (Novagen) using techniquesknown in the art such as described in Example 3, above.

To test their immunoreactivity, these truncated fragments of p23 andgp18 proteins were used in ELISA with sera from 6 BDV infected rats (15weeks post infection) and 7 immunoreactive schizophrenic patients (ofSection I above). Horse sera were only from acutely infected animals, ata stage of disease where antibodies to gp18 are not present, thus, serafrom 4 BDV infected horses were used only to study p23. For truncatedfragments of p23, similar patterns of immunoreactivity were found forsera from BDV infected rats, BDV infected horses and schizophrenicpatients. In the case of truncated fragments of unglycosylated gp18proteins, instead of using sera from BDV infected horses, the sera from2 mice immunized with native gp18 were tested (15 weeks postimmunization). The immunized mice were used to determine whether thetruncated fragments derived from unglycosylated gp18 were specificallyimmunoreactive with antibodies raised against native gp18. Again,similar patterns of immunoreactivity were found for sera from the BDVinfected rats, mice immunized with native gp18, and schizophrenicpatients. The above results are shown in FIGS. 20A and 21A,respectively, the taller blocks in the histograms indicate increasedimmunoreactivity relative to the shorter blocks. Significantly, theabove truncated fragments did not immunoreact with sera from the samecontrols (and the same number of controls) used in Section I above,whereas some sera from human controls with no neuropsychiatric diseaseimmunoreacted with the full length p23 and gp18 proteins (see Table 6).Thus, these fragments are more specific for detecting neuropsychiatricdisease than the full length proteins.

III. Epitope Mapping of Peptides Derived from p23 and gp18

Fine-mapping of epitopes with overlapping peptides of p23 and gp18 alsorevealed that the same determinants were detected by the above sera. Todetermine where an epitope was within each protein, a series ofoverlapping peptides were chemically synthesized and each peptide wastested for its ability to bind the antibody from schizophrenics and thesera of animals infected with BDV and/or immunized with p23 and gp18.

As shown in FIGS. 22 and 23, peptides of 8-mers were chemicallysynthesized, starting from the amino terminus of p23 and gp18 andspanning the full length of the proteins. Except for the peptides at theamino and carboxyl termini of p23 and gp18, each of the interveningpeptide overlaps its neighboring peptides (at its amino and carboxylends, respectively) by 4 amino acids.

To map the immunoepitopes on p23, the above 8-mer peptides derived fromrecp23 were tested against sera from: 6 rats infected with BDV (15 weekspost infection, p.I.); 2 rabbits immunized with recp23 (15 weeks postimmunization); and 7 immunoreactive schizophrenic patients (of Section Iabove). To map the immunoepitopes on gp18, the above 8-mer peptidesderived from unglycosylated recp18 were similarly tested, except thatthe rabbit sera were replaced with sera from 2 mice immunized withnative gp18 (15 weeks post immunization). The immunized mice were usedto determine whether the series of overlapping 8-mer peptides derivedfrom unglycosylated gp18 were specifically immunoreactive withantibodies raised against native gp18. The controls in both tests werethe same as in Section I above.

The tests were conducted using SPOTs membrane (Genosys Biotechnologies,Inc., The Woodlands, Tex., USA) and the technique described in Frank,R., et al., Tetrahedron, 44:6031-6040 (1988); Blankenmeyer-Menge, B., etal., Tetrahedron Letters, 29(46):5871-5874 (1988); Blankenmeyer-Menge,B., et al., in "Innovation and Perspectives in Solid-Phase Synthesis",(Epton, R. ed.), Chapman and Hall publ. (1989); and Blankenmeyer-Menge,B., et al., Tetrahedron Letters, 32(12):1701-1704 (1990) (the tests arehereinafter described as the "SPOTs tests"). The results are shown inFIGS. 22 and 23. The blocks in the histogram in each figure indicate thepeptides which immunoreact with the sera, the taller block indicatesincreased immunoreactivity relative to the shorter blocks. Based on theimmunoreactivity, the sequences of the epitopes were deduced. The aminoacid sequences of the epitopes and thus the peptides are as shown inTables 7 and 8, below, wherein the peptide sequences are shown from theamino terminus (left) to the carboxyl terminus (right):

                  TABLE 7                                                         ______________________________________                                        Peptides derived from p23                                                     ______________________________________                                        MATRPSSL         SEQ ID No. 20                                                NALTQPVDQLLK     SEQ ID No. 21                                                DQPTGREQ         SEQ ID No. 22                                                VRGTLGDI         SEQ ID No. 23                                                TAQRCDHS         SEQ ID No. 24                                                METMKLMMEKVD     SEQ ID No. 25                                                PMLPSHPA         SEQ ID No. 26                                                TADEWDII         SEQ ID No. 27                                                ______________________________________                                    

                  TABLE 8                                                         ______________________________________                                        Peptides derived from gp18                                                    ______________________________________                                        MNSKHSYV        SEQ ID No. 28                                                 TLMLEIDF        SEQ ID No. 29                                                 GHSLVNIYFQID    SEQ ID No. 30                                                 YKDPIRKY        SEQ ID No. 31                                                 AFNVFSYR        SEQ ID No. 32                                                 ______________________________________                                    

The result of representative SPOTs tests with the 8-mer peptides derivedfrom unglycosylated gp18 are graphically shown in FIG. 24A, the panelscontained sera from: 1 mouse immunized with native gp18, 1 rat infectedwith BDV, and 1 schizophrenic human, respectively. Each spot on thepanels indicates the immunoreation of a serum sample with an 8-merunglycosylated gp18 peptide. As shown in the scale on FIG. 24B, thedarker the spots, the higher the immunoreactivity. The lightest spot(Scale 1) indicates no detectable immunoreactivity; and the darkest spot(Scale 4) indicates highest immunoreactivity. As shown in FIG. 24A, theimmunoreactivity pattern of the sera against the peptides were similarfor all the animals/humans tested. Based on the pattern ofimmunoreactivity as shown by the spots, the epitopes E1 to E5 weremapped. The result of the epitope mapping is graphically shown in FIG.24B, the height of the blocks is directly proportional to the degree ofimmunoreactivity of the peptides tested which span the full length ofgp18, from amino acid at position 1 to position 142. The sequences ofthe mapped epitopes, E1 to E5, are listed below the histogram of FIG.24B. The epitopes mapped are the same as in Table 8, above, andconfirmed that the sera were specifically immunoreactive with epitopesfound within gp18. Again, significantly, the control sera did notimmunoreact with the peptides.

The same test was applied to the overlapping 8-mer peptides derived fromp23, except that the mice were immunized with recp23. A similar resultwas obtained, i.e. the immunoreactivity pattern of the sera against thepeptides were similar for all the animals/humans tested, and the testproduced the epitopes shown in Table 7, above. Again, significantly, thecontrol sera did not immunoreact with the peptides.

In summary, the above truncated fragments, epitopes and peptides, andnucleotide sequences which encode them or which are complementary tothese encoding nucleotide sequences, can be used to: (1) diagnose,prognose, monitor, and manage BDV infection/disease and schizophrenia,and more generally neurologic and neuropsychiatric diseases; and (2)vaccinate an animal or human against the foregoing infection anddiseases. Other useful truncated immunoreactive fragments, epitopes andpeptides can be similarly derived from the other BDV proteins using themethod of this Example. Thus, the nucleotide sequences encoding thesetruncated fragments, epitopes and peptides, nucleotide sequencescomplementary to the foregoing, and recombinant vectors and cellsexpressing the truncated fragments, epitopes and peptides, and theiruses are also claimed here. The vaccines, diagnostic, prognostic andmonitoring methods, recombinant vectors and cells, and nucleotidesequences, can be made using the teaching contained in this patentapplication in combination with methods known in the art. The abovefindings also suggest an association between BDV infection andschizophrenia.

All publications and patent applications mentioned in this Specificationare herein incorporated by reference to the same extent as if each ofthem had been individually indicated to be incorporated by reference.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity andunderstanding, it will be obvious that various modifications and changeswhich are within the skill of those skilled in the art are considered tofall within the scope of the appended claims. Future technologicaladvancements which allows for obvious changes in the basic inventionherein are also within the claims.

Deposit

The cDNA of BDV genomic RNA sequence has been deposited in the GenBankdata base (accession no. U04608). This GenBank sequence is herebyincorporated by reference in its entirety.

The recombinant transfer vector, suitable for transformation intoEscherichia coli DH10, containing four overlapping cDNA libraries (asdescribed in Example 1, above) representing the entire BDV viral genomehas been deposited under the Budapest Treaty, at the American TypeCulture Collection, Rockville, Md. 20852 (U.S.A.) on Dec. 30, 1994 underthe deposit name BDVU04608, and ATCC Accession No. 97008.

Availability of the deposited recombinant tranfer vector is not to beconstrued as a license to practice the invention in contravention of therights granted under the authority of any government in accordance withits patent laws.

Also, the present invention is not to be considered limited in scope bythe deposited recombinant transfer vector, since the deposited vector isintended only to be illustrative of particular aspects of the invention.Any recombinant transfer vector which can be used to prepare recombinantmicroorganism which can function to produce a recombinant proteinproduct described herein is considered to be within the scope of thisinvention. Further, various modifications of the invention in additionto those shown and described herein which are apparent to those skilledin the art from the preceding description are considered to fall withinthe scope of the appended claims.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                - (1) GENERAL INFORMATION:                                                    -    (iii) NUMBER OF SEQUENCES: 59                                            - (2) INFORMATION FOR SEQ ID NO:1:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 1112 base                                                         (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA to mRNA                                        -    (iii) HYPOTHETICAL: NO                                                   -     (iv) ANTI-SENSE: NO                                                     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                 - ATGCCACCCA AGAGACGCCT GGTTGATGAC GCCGATGCCA TGGAGGATCA AG - #ATCTATAT         60                                                                          - GAACCCCCAG CGAGCCTCCC TAAGCTCCCT GGGAAATTCC TACAATACAC CG - #TTGGGGGG        120                                                                          - TCTGACCCGC ATCCGGGTAT AGGGCATGAG AAAGACATCA GGCAGAACGC AG - #TGGCATGT        180                                                                          - TTAGACCAGT CACGGCGCGA TATGTTTCAC ACAGTAACGC CTAGCCTTGT GT - #TTCTATGT        240                                                                          - TTGCTAATCC CAGGACTGCA CGCTGCGTTT GTTCACGGAG GGGTGCCTCG TG - #AATCCTAC        300                                                                          - CTGTCGACGC CTGTCACGCG TGGAGAACAG ACTGTTGTTA AGACTGCGAA GT - #TTTACGGG        360                                                                          - GAAAAGACGA CGCAGCGTGA TCTCACCGAG CTGGAGATCT CCTCTATCTT CA - #GCCATTGT        420                                                                          - TGCTCATTAC TAATAGGGGT TGTGATAGGA TCGTCGTCTA AGATCAAAGC AG - #GAGCCGAG        480                                                                          - CAGATCAAGA AAAGGTTTAA AACTATGATG GCAGCCTTAA ACCGGCCATC CC - #ATGGTGAG        540                                                                          - ACTGCTACAC TACTCCAGAT GTTTAATCCA CATGAGGCTA TAGATTGGAT TA - #ACGGCCAA        600                                                                          - CCCTGGGTAG GCTCCTTTGT GTTGTCTCTA CTAACTACAG ACTTTGAGTC CC - #CAGGTAAA        660                                                                          - GAATTTATGG ACCAGATTAA GCTTGTCGCA AGTTATGCAC AGATGACTAC GT - #ACACTACT        720                                                                          - ATAAAGGAGT ACCTCGCAGA ATGCATGGAT GCTACCCTTA CAATCCCCGT AG - #TTGCATAT        780                                                                          - GAGATCCGTG ACTTTTTAGA AGTTTCAGCA AAGCTTAAGG AGGATCATGC TG - #ACCTGTTC        840                                                                          - CCGTTTCTGG GGGCCATTAG ACACCCCGAC GCTATCAAGC TGGCGCCACG AA - #GCTTTCCC        900                                                                          - AATCTGGCCT CCGCAGCGTT TTACTGGAGT AAGAAGGAAA ACCCCACAAT GG - #CAGGCTAC        960                                                                          - CGGGCCTCCA CCATCCAGCC GGGCGCAAGT GTCAAGGAAA CCCAGCTTGC CC - #GGTATAGG       1020                                                                          - CGCCGCGAGA TATCTCGTGG AGAGGACGGG GCAGAGCTCT CAGGTGAGAT CT - #CTGCCATA       1080                                                                          #        1112      TGAC TGGTCTAAAC TA                                         - (2) INFORMATION FOR SEQ ID NO:2:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 370 amino                                                         (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: unknown                                               -     (ii) MOLECULE TYPE: protein                                             -    (iii) HYPOTHETICAL: NO                                                   -     (iv) ANTI-SENSE: NO                                                     -      (v) FRAGMENT TYPE: internal                                            -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                 - Met Pro Pro Lys Arg Arg Leu Val Asp Asp Al - #a Asp Ala Met Glu Asp         #                15                                                           - Gln Asp Leu Tyr Glu Pro Pro Ala Ser Leu Pr - #o Lys Leu Pro Gly Lys         #            30                                                               - Phe Leu Gln Tyr Thr Val Gly Gly Ser Asp Pr - #o His Pro Gly Ile Gly         #        45                                                                   - His Glu Lys Asp Ile Arg Gln Asn Ala Val Al - #a Leu Leu Asp Gln Ser         #    60                                                                       - Arg Arg Asp Met Phe His Thr Val Thr Pro Se - #r Leu Val Phe Leu Cys         #80                                                                           - Leu Leu Ile Pro Gly Leu His Ala Ala Phe Va - #l His Gly Gly Val Pro         #                95                                                           - Arg Glu Ser Tyr Leu Ser Thr Pro Val Thr Ar - #g Gly Glu Gln Thr Val         #           110                                                               - Val Lys Thr Ala Lys Phe Tyr Gly Glu Lys Th - #r Thr Gln Arg Asp Leu         #       125                                                                   - Thr Glu Leu Glu Ile Ser Ser Ile Phe Ser Hi - #s Cys Cys Ser Leu Leu         #   140                                                                       - Ile Gly Val Val Ile Gly Ser Ser Ser Lys Il - #e Lys Ala Gly Ala Glu         145                 1 - #50                 1 - #55                 1 -       #60                                                                           - Gln Ile Lys Lys Arg Phe Lys Thr Met Met Al - #a Ala Leu Asn Arg Pro         #               175                                                           - Ser His Gly Glu Thr Ala Thr Leu Leu Gln Me - #t Phe Asn Pro His Glu         #           190                                                               - Ala Ile Asp Trp Ile Asn Gly Gln Pro Trp Va - #l Gly Ser Phe Val Leu         #       205                                                                   - Ser Leu Leu Thr Thr Asp Phe Glu Ser Pro Gl - #y Lys Glu Phe Met Asp         #   220                                                                       - Gln Ile Lys Leu Val Ala Ser Tyr Ala Gln Me - #t Thr Thr Tyr Thr Thr         225                 2 - #30                 2 - #35                 2 -       #40                                                                           - Ile Lys Glu Tyr Leu Ala Glu Cys Met Asp Al - #a Thr Leu Thr Ile Pro         #               255                                                           - Val Val Ala Tyr Glu Ile Arg Asp Phe Leu Gl - #u Val Ser Ala Lys Leu         #           270                                                               - Lys Glu Asp His Ala Asp Leu Phe Pro Phe Le - #u Gly Ala Ile Arg His         #       285                                                                   - Pro Asp Ala Ile Lys Leu Ala Pro Arg Ser Ph - #e Pro Asn Leu Ala Ser         #   300                                                                       - Ala Ala Phe Tyr Trp Ser Lys Lys Glu Asn Pr - #o Thr Met Ala Gly Tyr         305                 3 - #10                 3 - #15                 3 -       #20                                                                           - Arg Ala Ser Thr Ile Gln Pro Gly Ala Ser Va - #l Lys Glu Thr Gln Leu         #               335                                                           - Ala Arg Tyr Arg Arg Arg Glu Ile Ser Arg Gl - #y Glu Asp Gly Ala Glu         #           350                                                               - Leu Ser Gly Glu Ile Ser Ala Ile Met Lys Me - #t Ile Gly Val Thr Gly         #       365                                                                   - Leu Asn                                                                         370                                                                       - (2) INFORMATION FOR SEQ ID NO:3:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 609 base                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA to mRNA                                        -    (iii) HYPOTHETICAL: NO                                                   -     (iv) ANTI-SENSE: NO                                                     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                 - ATGGCAACGC GACCATCGAG TCTGGTCGAC TCCCTGGAGG ACGAAGAAGA TC - #CCCAGACA         60                                                                          - CTACGACGGG AACGACCGGG GTCACCAAGA CCACGGAAGG TCCCAAGGAA TG - #CATTGACC        120                                                                          - CAACCAGTAG ACCAGCTCCT GAAGGACCTC AGGAAGAACC CCTCCATGAT CT - #CAGACCCA        180                                                                          - GACCAGCGAA CCGGAAGGGA GCAGCTGTCG AATGATGAGC TAATCAAGAA GT - #TAGTGACG        240                                                                          - GAGCTGGCCG AGAATAGCAT GATCGAGGCT GAGGAGGTGC GGGGCACTCT TG - #GAGACATC        300                                                                          - TCGGCTCGTA TCGAGGCAGG GTTTGAGTCC CTGTCCGCCC TCCAAGTGGA AA - #CCATCCAG        360                                                                          - ACAGCTCAGC GGTGCGATCA CTCCGACAGC ATCAGGATCC TCGGCGAGAA CA - #TCAAGATA        420                                                                          - CTAGATCGCT CCATGAAGAC AATGATGGAG ACAATGAAGC TCATGATGGA GA - #AGGTGGAT        480                                                                          - CTCCTCTACG CATCAACCGC CGTTGGGACC TCTGCACCCA TGTTGCCCTC CC - #ATCCTGCA        540                                                                          - CCTCCGCGCA TTTATCCCCA GCTCCCAAGT GCCCCGACAA CGGATGAATG GG - #ACATCATA        600                                                                          #        609                                                                  - (2) INFORMATION FOR SEQ ID NO:4:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 201 amino                                                         (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: unknown                                               -     (ii) MOLECULE TYPE: protein                                             -    (iii) HYPOTHETICAL: NO                                                   -     (iv) ANTI-SENSE: NO                                                     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                 - Met Ala Thr Arg Pro Ser Ser Leu Val Asp Se - #r Leu Glu Asp Glu Glu         #                15                                                           - Asp Pro Gln Thr Leu Arg Arg Glu Arg Pro Gl - #y Ser Pro Arg Pro Arg         #            30                                                               - Lys Val Pro Arg Asn Ala Leu Thr Gln Pro Va - #l Asp Gln Leu Leu Lys         #        45                                                                   - Asp Leu Arg Lys Asn Pro Ser Met Ile Ser As - #p Pro Asp Gln Arg Thr         #    60                                                                       - Gly Arg Glu Gln Leu Ser Asn Asp Glu Leu Il - #e Lys Lys Leu Val Thr         #80                                                                           - Glu Leu Ala Glu Asn Ser Met Ile Glu Ala Gl - #u Glu Val Arg Gly Thr         #                95                                                           - Leu Gly Asp Ile Ser Ala Arg Ile Glu Ala Gl - #y Phe Glu Ser Leu Ser         #           110                                                               - Ala Leu Gln Val Glu Thr Ile Gln Thr Ala Gl - #n Arg Cys Asp His Ser         #       125                                                                   - Asp Ser Ile Arg Ile Leu Gly Glu Asn Ile Ly - #s Ile Leu Asp Arg Ser         #   140                                                                       - Met Lys Thr Met Met Glu Thr Met Lys Leu Me - #t Met Glu Lys Val Asp         145                 1 - #50                 1 - #55                 1 -       #60                                                                           - Leu Leu Tyr Ala Ser Thr Ala Val Gly Thr Se - #r Ala Pro Met Leu Pro         #               175                                                           - Ser His Pro Ala Pro Pro Arg Ile Tyr Pro Gl - #n Leu Pro Ser Ala Pro         #           190                                                               - Thr Thr Asp Glu Trp Asp Ile Ile Pro                                         #       200                                                                   - (2) INFORMATION FOR SEQ ID NO:5:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 428 base                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA to mRNA                                        -    (iii) HYPOTHETICAL: NO                                                   -     (iv) ANTI-SENSE: NO                                                     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                 - ATGAATTCAA AACATTCCTA TGTGGAGCTC AAGGACAAGG TAATCGTCCC TG - #GATGGCCC         60                                                                          - ACACTGATGC TTGAGATAGA CTTTGTAGGG GGGACTTCAC GGAACCAGTT CC - #TTAACATC        120                                                                          - CCATTTCTTT CAGTGAAAGA GCCTCTGCAG CTTCCACGCG AGAAGAAGTT GA - #CCGACTAC        180                                                                          - TTTACTATTG ACGTAGAACC AGCAGGTCAT TCCCTGGTCA ATATATACTT CC - #AGATTGAC        240                                                                          - GACTTCTTGC TCCTAACACT CAACTCACTA TCTGTGTACA AGGACCCGAT TA - #GAAAATAC        300                                                                          - ATGTTCCTAC GCCTCAACAA GGACCAGAGC AAGCACGCAA TCAATGCAGC CT - #TCAATGTC        360                                                                          - TTTTCTTATC GGCTTCGGAA CATTGGTGTT GGTCCTCTCG GCCCGGACAT TC - #GATCTTCA        420                                                                          #         428                                                                 - (2) INFORMATION FOR SEQ ID NO:6:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 142 amino                                                         (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: unknown                                               -     (ii) MOLECULE TYPE: protein                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                 - Met Asn Ser Lys His Ser Tyr Val Glu Leu Ly - #s Asp Lys Val Ile Val         #                15                                                           - Pro Gly Trp Pro Thr Leu Met Leu Glu Ile As - #p Phe Val Gly Gly Thr         #            30                                                               - Ser Arg Asn Gln Phe Leu Asn Ile Pro Phe Le - #u Ser Val Lys Glu Pro         #        45                                                                   - Leu Gln Leu Pro Arg Glu Lys Lys Leu Thr As - #p Tyr Phe Thr Ile Asp         #    60                                                                       - Val Glu Pro Ala Gly His Ser Leu Val Asn Il - #e Tyr Phe Gln Ile Asp         #80                                                                           - Asp Phe Leu Leu Leu Thr Leu Asn Ser Leu Se - #r Val Tyr Lys Asp Pro         #                95                                                           - Ile Arg Lys Tyr Met Phe Leu Arg Leu Asn Ly - #s Asp Gln Ser Lys His         #           110                                                               - Ala Ile Asn Ala Ala Phe Asn Val Phe Ser Ty - #r Arg Leu Arg Asn Ile         #       125                                                                   - Gly Val Gly Pro Leu Gly Pro Asp Ile Arg Se - #r Ser Gly Pro                 #   140                                                                       - (2) INFORMATION FOR SEQ ID NO:7:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 1515 base                                                         (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA to mRNA                                        -    (iii) HYPOTHETICAL: NO                                                   -     (iv) ANTI-SENSE: NO                                                     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                 - ATGCAGCCTT CAATGTCTTT TCTTATCGGC TTCGGAACAT TGGTGTTGGT CC - #TCTCGGCC         60                                                                          - CGGACATTCG ATCTTCAGGG CCTTAGCTGC AATACTGACT CCACTCCTGG AC - #TGATTGAC        120                                                                          - CTGGAGATAA GGCGACTTTG CCACACCCCA ACGGAAAATG TCATTTCATG CG - #AGGTTAGT        180                                                                          - TATCTCAACC ACACGACTAT TAGCCTCCCG GCAGTCCACA CATCATGCCT CA - #AGTACCAC        240                                                                          - TGCAAAACCT ATTGGGGATT CTTTGGTAGC TACAGCGCTG ACCGAATCAT AA - #ATCGGTAC        300                                                                          - ACTGGTACTG TTAAGGGTTG TCTAAACAAC TCAGCACCAG AGGACCCCTT CG - #AGTGCAAC        360                                                                          - TGGTTCTACT GCTGCTCGGC GATTACAACA GAGATCTGCC GATGCTCTAT TA - #CAAATGTC        420                                                                          - ACGGTGGCTG TGCAAACATT CCCACCGTTC ATGTACTGCA GTTTTGCAGA CT - #GCAGTACC        480                                                                          - GTGAGCCAAC AGGAGCTAGA GAGTGGAAAG GCAATGCTGA GCGATGGCAG TA - #CATTAACT        540                                                                          - TATACCCCGT ATATCCTACA GTCAGAAGTC GTGAACAAAA CCCTCAATGG GA - #CCATACTC        600                                                                          - TGCAACTCAT CCTCTAAGAT AGTTTCCTTC GATGAATTTA GGCGTTCATA CT - #CCCTAACG        660                                                                          - AATGGTAGTT ACCAGAGCTC ATCAATCAAT GTGACGTGTG CAAACTACAC GT - #CGTCCTGC        720                                                                          - CGGCCCAGGT TGAAAAGGCG GCGTAGGGAC ACCCAGCAGA TTGAGTATCT AG - #TTCACAAG        780                                                                          - CTTAGGCCCA CACTGAAAGA TGCATGGGAG GACTGTGAGA TCCTCCAGTC TC - #TGCTCCTA        840                                                                          - GGGGTGTTTG GTACTGGGAT CGCAAGTGCT TCTCAATTTT TGAGGAGCTG GC - #TCAACCAC        900                                                                          - CCTGACATCA TCGGGTATAT AGTTAATGGA GTTGGGGTTG TCTGGCAATG CC - #ATCGTGTT        960                                                                          - AATGTCACGT TCATGGCGTG GAATGAGTCC ACCTATTACC CTCCAGTAGA TT - #ACAATGGG       1020                                                                          - CGGAAGTACT TCCTGAATGA TGAGGGAAGG TTACAAACAA ACACCCCCGA GG - #CAAGGCCA       1080                                                                          - GGGCTTAAGC GGGTCATGTG GTTCGGCAGG TACTTCCTAG GGACAGTAGG GT - #CTGGGGTG       1140                                                                          - AAACCGAGGA GGATTCGGTA CAATAAGACC TCACATGACT ACCACCTGGA GG - #AGTTTGAG       1200                                                                          - GCAAGTCTCA ACATGACCCC TCAGACCAGT ATCGCCTCGG GTCATGAGAC AG - #ACCCCATA       1260                                                                          - AATCATGCCT ACGGAACGCA GGCTGATCTC CTTCCATACA CCAGGTCTAG TA - #ATATAACA       1320                                                                          - TCTACGGATA CAGGCTCAGG CTGGGTGCAC ATCGGCCTAC CCTCATTTGC TT - #TCCTCAAT       1380                                                                          - CCCCTCGGGT GGCTCAGGGA CCTACTTGCA TGGGCAGCCT GGTTGGGTGG GG - #TTCTATAC       1440                                                                          - TTAATAAGTC TTTGTGTTTC CTTACCAGCC TCCTTCGCGA GGAGGAGACG CC - #TCGGCCGG       1500                                                                          #  1515                                                                       - (2) INFORMATION FOR SEQ ID NO:8:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 503 amino                                                         (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: unknown                                               -     (ii) MOLECULE TYPE: protein                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                 - Met Gln Pro Ser Met Ser Phe Leu Ile Gly Ph - #e Gly Thr Leu Val Leu         #                15                                                           - Val Leu Ser Ala Arg Thr Phe Asp Leu Gln Gl - #y Leu Ser Cys Asn Thr         #            30                                                               - Asp Ser Thr Pro Gly Leu Ile Asp Leu Glu Il - #e Arg Arg Leu Cys His         #        45                                                                   - Thr Pro Thr Glu Asn Val Ile Ser Cys Glu Va - #l Ser Tyr Leu Asn His         #    60                                                                       - Thr Thr Ile Ser Leu Pro Ala Val His Thr Se - #r Cys Leu Lys Tyr His         #80                                                                           - Cys Lys Thr Tyr Trp Gly Phe Phe Gly Ser Ty - #r Ser Ala Asp Arg Ile         #                95                                                           - Ile Asn Arg Tyr Thr Gly Thr Val Lys Gly Cy - #s Leu Asn Asn Ser Ala         #           110                                                               - Pro Glu Asp Pro Phe Glu Cys Asn Trp Phe Ty - #r Cys Cys Ser Ala Ile         #       125                                                                   - Thr Thr Glu Ile Cys Arg Cys Ser Ile Thr As - #n Val Thr Val Ala Val         #   140                                                                       - Gln Thr Phe Pro Pro Phe Met Tyr Cys Ser Ph - #e Ala Asp Cys Ser Thr         145                 1 - #50                 1 - #55                 1 -       #60                                                                           - Val Ser Gln Gln Glu Leu Glu Ser Gly Lys Al - #a Met Leu Ser Asp Gly         #               175                                                           - Ser Thr Leu Thr Tyr Thr Pro Tyr Ile Leu Gl - #n Ser Glu Val Val Asn         #           190                                                               - Lys Thr Leu Asn Gly Thr Ile Leu Cys Asn Se - #r Ser Ser Lys Ile Val         #       205                                                                   - Ser Phe Asp Glu Phe Arg Arg Ser Tyr Ser Le - #u Thr Asn Gly Ser Tyr         #   220                                                                       - Gln Ser Ser Ser Ile Asn Val Thr Cys Ala As - #n Tyr Thr Ser Ser Cys         225                 2 - #30                 2 - #35                 2 -       #40                                                                           - Arg Pro Arg Leu Lys Arg Arg Arg Arg Asp Th - #r Gln Gln Ile Glu Tyr         #               255                                                           - Leu Val His Lys Leu Arg Pro Thr Leu Lys As - #p Ala Trp Glu Asp Cys         #           270                                                               - Glu Ile Leu Gln Ser Leu Leu Leu Gly Val Ph - #e Gly Thr Gly Ile Ala         #       285                                                                   - Ser Ala Ser Gln Phe Leu Arg Ser Trp Leu As - #n His Pro Asp Ile Ile         #   300                                                                       - Gly Tyr Ile Val Asn Gly Val Gly Val Val Tr - #p Gln Cys His Arg Val         305                 3 - #10                 3 - #15                 3 -       #20                                                                           - Asn Val Thr Phe Met Ala Trp Asn Glu Ser Th - #r Tyr Tyr Pro Pro Val         #               335                                                           - Asp Tyr Asn Gly Arg Lys Tyr Phe Leu Asn As - #p Glu Gly Arg Leu Gln         #           350                                                               - Thr Asn Thr Pro Glu Ala Arg Pro Gly Leu Ly - #s Arg Val Met Trp Phe         #       365                                                                   - Gly Arg Tyr Phe Leu Gly Thr Val Gly Ser Gl - #y Val Lys Pro Arg Arg         #   380                                                                       - Ile Arg Tyr Asn Lys Thr Ser His Asp Tyr Hi - #s Leu Glu Glu Phe Glu         385                 3 - #90                 3 - #95                 4 -       #00                                                                           - Ala Ser Leu Asn Met Thr Pro Gln Thr Ser Il - #e Ala Ser Gly His Glu         #               415                                                           - Thr Asp Pro Ile Asn His Ala Tyr Gly Thr Gl - #n Ala Asp Leu Leu Pro         #           430                                                               - Tyr Thr Arg Ser Ser Asn Ile Thr Ser Thr As - #p Thr Gly Ser Gly Trp         #       445                                                                   - Val His Ile Gly Leu Pro Ser Phe Ala Phe Le - #u Asn Pro Leu Gly Trp         #   460                                                                       - Leu Arg Asp Leu Leu Ala Trp Ala Ala Trp Le - #u Gly Gly Val Leu Tyr         465                 4 - #70                 4 - #75                 4 -       #80                                                                           - Leu Ile Ser Leu Cys Val Ser Leu Pro Ala Se - #r Phe Ala Arg Arg Arg         #               495                                                           - Arg Leu Gly Arg Trp Gln Glu                                                             500                                                               - (2) INFORMATION FOR SEQ ID NO:9:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 5135 base                                                         (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA to mRNA                                        -    (iii) HYPOTHETICAL: NO                                                   -     (iv) ANTI-SENSE: NO                                                     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                 - ATGTCATTTC ATGCGAGCCT CCTTCGCGAG GAGGAGACGC CTCGGCCGGT GG - #CAGGAATA         60                                                                          - AACCGTACCG ACCAGTCTCT TAAAAACCCT CTCCTCGGAA CAGAGGTCTC TT - #TCTGCCTT        120                                                                          - AAGTCGAGCT CACTCCCCCA TCATGTACGA GCACTAGGCC AGATTAAAGC AA - #GGAACCTG        180                                                                          - GCATCCTGTG ACTATTACTT GCTATTCCGC CAAGTTGTAT TGCCCCCTGA AG - #TATATCCC        240                                                                          - ATTGGTGTTC TAATAAGAGC TGCGGAGGCT ATACTAACAG TTATAGTATC AG - #CTTGGAAG        300                                                                          - CTGGATCATA TGACGAAGAC CCTATACTCC TCTGTGAGAT ATGCACTCAC CA - #ATCCCCGG        360                                                                          - GTCCGAGCCC AACTTGAGCT TCACATTGCC TACCAGCGCA TAGTGGGTCA GG - #TCTCGTAC        420                                                                          - AGCCGGGAGG CAGACATAGG GCCAAAAAGG CTTGGGAATA TGTCATTGCA AT - #TCATCCAA        480                                                                          - TCTCTCGTTA TTGCCACCAT AGACACGACA AGCTGCCTAA TGACCTACAA CC - #ACTTTCTT        540                                                                          - GCTGCAGCAG ACACAGCCAA GAGCAGATGC CATCTCCTAA TCGCCTCAGT GG - #TCCAGGGG        600                                                                          - GCCCTTTGGG AACAAGGGTC ATTTCTTGAT CATATAATCA ACATGATCGA CA - #TAATTGAC        660                                                                          - TCAATCAACC TCCCCCATGA TGATTACTTC ACAATTATTA AGTCTATCTT TC - #CCTACTCC        720                                                                          - CAAGGGCTTG TTATGGGGAG GCATAATGTA TCAGTCTCCT CTGATTTCGC GT - #CCGTATTT        780                                                                          - GCCATTCCTG AATTATGCCC GCAACTAGAC AGCTTACTAA AAAAACTGCT CC - #AACTTGAC        840                                                                          - CCCGTTCTCC TCCTCATGGT CTCTTCGGTG CAGAAGTCAT GGTACTTCCC TG - #AGATCCGA        900                                                                          - ATGGTCGACG GGTCACGGGA GCAGCTCCAC AAGATGCGTG TCGAGCTGGA AA - #CGCCCCAA        960                                                                          - GCCCTGCTGT CGTACGGCCA TACCCTCCTG TCAATATTTC GGGCAGAGTT TA - #TCAAAGGC       1020                                                                          - TATGTCTCAA AGAATGCGAA GTGGCCGCCC GTACACCTGC TCCCAGGCTG TG - #ACAAATCC       1080                                                                          - ATAAAAAATG CGAGAGAGCT GGGCCGCTGG AGCCCGGCAT TTGACCGACG AT - #GGCAGCTC       1140                                                                          - TTCGAGAAGG TTGTCATTCT AAGAATTGCT GACCTAGATA TGGATCCCGA CT - #TCAACGAT       1200                                                                          - ATTGTTAGCG ATAAGGCGAT AATCAGCTCA AGAAGGGACT GGGTATTCGA GT - #ACAATGCA       1260                                                                          - GCGGCCTTTT GGAAGAAATA CGGTGAACGG TTGGAGAGGC CTCCTGCCAG GT - #CGGGACCG       1320                                                                          - TCACGACTTG TGAATGCTCT AATCGATGGA CGCTTAGACA ATATCCCAGC CC - #TGCTAGAG       1380                                                                          - CCATTTTACA GGGGAGCGGT TGAGTTCGAG GATCGGTTGA CTGTGCTCGT GC - #CTAAGGAG       1440                                                                          - AAAGAGTTAA AGGTAAAGGG AAGGTTCTTC TCGAAGCAAA CATTGGCAAT CA - #GGATATAT       1500                                                                          - CAGGTTGTTG CTGAAGCTGC ACTTAAGAAT GAGGTTATGC CATACCTAAA GA - #CACACTCA       1560                                                                          - ATGACCATGA GCTCAACGGC TCTAACTCAC CTTCTTAACC GGCTATCACA TA - #CTATCACT       1620                                                                          - AAGGGTGACT CCTTTGTTAT TAACCTTGAC TATAGTTCCT GGTGCAACGG TT - #TCCGACCA       1680                                                                          - GAACTGCAGG CCCCAATCTG TCGTCAGTTG GATCAGATGT TCAATTGCGG GT - #ACTTCTTC       1740                                                                          - AGGACTGGGT GCACACTGCC ATGCTTTACC ACGTTTATTA TTCAAGACAG GT - #TCAACCCG       1800                                                                          - CCCTATTCCC TCAGTGGTGA GCCCGTTGAA GACGGAGTTA CATGCGCGGT TG - #GGACTAAA       1860                                                                          - ACAATGGGGG AGGGCATGAG GCAGAAACTA TGGACAATCC TTACGAGCTG CT - #GGGAGATA       1920                                                                          - ATTGCTCTTC GGGAAATTAA CGTGACGTTT AACATACTAG GCCAAGGTGA TA - #ATCAGACA       1980                                                                          - ATCATCATAC ATAAATCTGC AAGCCAAAAT AACCAGCTAT TAGCGGAGCG AG - #CACTAGGG       2040                                                                          - GCCCTGTACA AGCATGCTAG ATTAGCTGGC CATAACCTCA AGGTAGAGGA AT - #GCTGGGTG       2100                                                                          - TCAGATTGTC TGTATGAGTA TGGAAAGAAG CTTTTCTTCC GTGGTGTACC TG - #TCCCGGGC       2160                                                                          - TGTTTGAAGC AGCTCTCACG GGTGACGGAT TCTACTGGAG AGCTATTCCC AA - #ACCTATAC       2220                                                                          - TCAAAGTTAG CCTGCTTAAC ATCATCGTGT TTAAGCGCAG CGATGGCAGA CA - #CATCTCCA       2280                                                                          - TGGGTGGCAC TCGCGACAGG TGTCTGTCTG TATCTTATCG AGTTATATGT TG - #AGCTGCCT       2340                                                                          - CCAGCAATCA TGCAGGATGA GTCGCTATTG ACGACCCTCT GCCTCGTAGG CC - #CATCCATT       2400                                                                          - GGTGGGCTTC CGACCCCTGC AACCCTACCC AGTGTCTTTT TCAGAGGAAT GT - #CCGACCCA       2460                                                                          - CTGCCCTTTC AGCTAGCACT CTTGCAGACC CTCATTAAGA CGACAGGGGT GA - #CCTGTAGC       2520                                                                          - TTGGTGAATC GTGTGGTCAA GTTACGGATA GCACCCTATC CAGACTGGCT CT - #CTCTAGTG       2580                                                                          - ACTGACCCGA CCTCACTCAA CATTGCCCAA GTGTACCGGC CAGAACGTCA GA - #TCAGGAGG       2640                                                                          - TGGATTGAGG AAGCGATAGC GACAAGCTCA CACTCGTCAC GCATAGCAAC TT - #TCTTCCAG       2700                                                                          - CAGCCCCTCA CGGAGATGGC TCAGTTGCTT GCGAGGGACC TTTCAACAAT GA - #TGCCTCTT       2760                                                                          - CGACCCCGGG ATATGTCGGC CTTATTCGCA TTATCAAATG TCGCATACGG TT - #TAAGCATT       2820                                                                          - ATAGATCTAT TTCAAAAATC CTCTACCGTT GTTTCTGCAA GTCAAGCTGT CC - #ATATCGAG       2880                                                                          - GATGTTGCCC TAGAGAGTGT AAGGTATAAG GAATCTATCA TCCAGGGTCT GT - #TAGACACC       2940                                                                          - ACTGAGGGGT ATAACATGCA ACCTTATTTG GAAGGTTGCA CTTACCTTGC AG - #CCAAACAG       3000                                                                          - TTACGTAGGT TGACATGGGG TCGAGACCTA GTTGGAGTCA CAATGCCGTT TG - #TTGCCGAG       3060                                                                          - CAATTCCATC CTCACAGTTC TGTGGGTGCA AAGGCGGAAC TCTACCTCGA CG - #CTATTATA       3120                                                                          - TACTGCCCAC AGGAGACATT GCGGTCACAC CATCTGACTA CCAGGGGGGA CC - #AGCCGCTT       3180                                                                          - TACCTCGGAT CCAATACGGC TGTCAAGGTC CAGCGAGGTG AGATCACGGG CC - #TAACAAAG       3240                                                                          - TCAAGGGCTG CAAATCTAGT CAGGGACACT CTCGTTCTCC ATCAGTGGTA TA - #AAGTCCGT       3300                                                                          - AAAGTTACCG ATCCACACTT GAACACCCTC ATGGCACGCT TCTTACTTGA GA - #AGGGGTAC       3360                                                                          - ACATCTGACG CTCGACCTAG CATCCAGGGT GGGACCCTCA CGCATCGTCT CC - #CATCCCGC       3420                                                                          - GGAGACTCAC GGCAGGGGCT TACTGGGTAT GTAAATATAC TAAGTACGTG GC - #TTCGATTC       3480                                                                          - TCAAGTGATT ATCTTCACTC TTTCTCGAAA TCATCAGACG ACTATACAAT CC - #ACTTTCAG       3540                                                                          - CATGTATTCA CATACGGTTG CCTCTATGCT GATTCGGTGA TTAGATCGGG CG - #GTGTTATT       3600                                                                          - TCCACTCCTT ACCTTTTGAG TGCAAGTTGT AAAACATGCT TTGAGAAGAT AG - #ACTCAGAG       3660                                                                          - GAGTTCGTCC TGGCATGTGA ACCCCAATAC AGGGGTGCTG AGTGGCTGAT AT - #CAAAGCCA       3720                                                                          - GTCACTGTCC CTGAGCAGAT AACTGATGCT GAAGTCGAGT TTGACCCCTG TG - #TGAGTGCG       3780                                                                          - GGTTATTGTC TCGGGATTCT CATTGGCAAG TCATTCTTAG TTGACATAAG GG - #CAAGTGGG       3840                                                                          - CATGATATCA TGGAGCAGCG GACATGGGCT AACCTGGAGA GGTTTTCTGT AT - #CGGACATG       3900                                                                          - CAGAAACTTC CGTGGAGTAT TGTAATTCGG TCTCTCTGGA GATTCCTTAT TG - #GCGCACGG       3960                                                                          - CTCCTTCAGT TTGAGAAGGC TGGCCTCATT AGAATGCTGT ATGCTGCGAC AG - #GTCCAACC       4020                                                                          - CCTAGCTTCC TAATGAAAGT TTTTCAAGAC TCAGCCCTCC TCATGGACTG CG - #CACCCCTC       4080                                                                          - GATCGGCTGT CCCCTAGGAT CAACTTTCAT AGTCGGGGAG ACCTCGTTGC TA - #AGCTTGTT       4140                                                                          - TTATTGCCCT TCATCAACCC GGGTATAGTG GAGATTGAAG TGTCTGGAAT TA - #ATAGCAAG       4200                                                                          - TACCATGCAG TATCGGAGGC CAATATGGAT CTGTACATCG CTGCTGCCAA GT - #CTGTGGGC       4260                                                                          - GTGAAGCCCA CACAGTTTGT TGAGGAAACA AACGACTTTA CGGCCCGCGG CC - #ACCACCAT       4320                                                                          - GGTTGTTATT CCCTTTCTTG GTCTAAGTCA CGCAATCAAT CACAGGTCCT AA - #AGATGGTA       4380                                                                          - GTACGGAAGC TGAAGCTCTG TGTCCTGTAT ATATACCCCA CAGTCGATCC CG - #CCGTTGCT       4440                                                                          - CTCGACCTGT GCCATCTACC AGCATTAACT ATAATCCTAG TGCTCGGCGG TG - #ACCCAGCG       4500                                                                          - TACTATGAGC GATTACTTGA GATGGACCTG TGCGGGGCTG TGTCAAGTCG AG - #TCGATATC       4560                                                                          - CCCCATTCTC TGGCTGGCAG AACGCACAGG GGGTTCGCAG TGGGCCCAGA CG - #CTGGTCCA       4620                                                                          - GGTGTAATTA GACTCGACAG GTTAGAGTCA GTTTGTTATG CTCACCCCTG TT - #TAGAGGAA       4680                                                                          - CTAGAGTTTA ATGCATATCT AGACTCTGAG TTGGTTGACA TTAGTGATAT GT - #GCTGCCTC       4740                                                                          - CCCTTAGCGA CACCCTGTAA GGCCCTTTTC AGGCCAATAT ATCGGAGCTT AC - #AGTCGTTC       4800                                                                          - AGGTTAGCCT TAATGGACAA CTATAGTTTT GTCATGGACC TCATTATGAT CC - #GAGGACTG       4860                                                                          - GACATTAGGC CTCACCTTGA GGAATTTGAC GAGCTGCTTG TGGTAGGACA GC - #ACATCCTC       4920                                                                          - GGCCAGCCCG TCCTAGTAGA GGTTGTTTAC TACGTTGGAG TTGTTAGGAA GC - #GCCCTGTG       4980                                                                          - TTAGCGAGGC ATCCGTGGTC AGCAGATCTT AAGCGAATTA CTGTGGGGGG GC - #GGGCTCCC       5040                                                                          - TGCCCCTCTG CTGCCAGATT GCGTGATGAG GATTGTCAGG GGTCTCTGTT GG - #TTGGGCTT       5100                                                                          #     5135         AGTT ATTGATAATT GATTA                                      - (2) INFORMATION FOR SEQ ID NO:10:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 1711 amino                                                        (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: unknown                                               -     (ii) MOLECULE TYPE: protein                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                - Met Ser Phe His Ala Ser Leu Leu Arg Glu Gl - #u Glu Thr Pro Arg Pro         #                15                                                           - Val Ala Gly Ile Asn Arg Thr Asp Gln Ser Le - #u Lys Asn Pro Leu Leu         #            30                                                               - Gly Thr Glu Val Ser Phe Cys Leu Lys Ser Se - #r Ser Leu Pro His His         #        45                                                                   - Val Arg Ala Leu Gly Gln Ile Lys Ala Arg As - #n Leu Ala Ser Cys Asp         #    60                                                                       - Tyr Tyr Leu Leu Phe Arg Gln Val Val Leu Pr - #o Pro Glu Val Tyr Pro         #80                                                                           - Ile Gly Val Leu Ile Arg Ala Ala Glu Ala Il - #e Leu Thr Val Ile Val         #                95                                                           - Ser Ala Trp Lys Leu Asp His Met Thr Lys Th - #r Leu Tyr Ser Ser Val         #           110                                                               - Arg Tyr Ala Leu Thr Asn Pro Arg Val Arg Al - #a Gln Leu Glu Leu His         #       125                                                                   - Ile Ala Tyr Gln Arg Ile Val Gly Gln Val Se - #r Tyr Ser Arg Glu Ala         #   140                                                                       - Asp Ile Gly Pro Lys Arg Leu Gly Asn Met Se - #r Leu Gln Phe Ile Gln         145                 1 - #50                 1 - #55                 1 -       #60                                                                           - Ser Leu Val Ile Ala Thr Ile Asp Thr Thr Se - #r Cys Leu Met Thr Tyr         #               175                                                           - Asn His Phe Leu Ala Ala Ala Asp Thr Ala Ly - #s Ser Arg Cys His Leu         #           190                                                               - Leu Ile Ala Ser Val Val Gln Gly Ala Leu Tr - #p Glu Gln Gly Ser Phe         #       205                                                                   - Leu Asp His Ile Ile Asn Met Ile Asp Ile Il - #e Asp Ser Ile Asn Leu         #   220                                                                       - Pro His Asp Asp Tyr Phe Thr Ile Ile Lys Se - #r Ile Phe Pro Tyr Ser         225                 2 - #30                 2 - #35                 2 -       #40                                                                           - Gln Gly Leu Val Met Gly Arg His Asn Val Se - #r Val Ser Ser Asp Phe         #               255                                                           - Ala Ser Val Phe Ala Ile Pro Glu Leu Cys Pr - #o Gln Leu Asp Ser Leu         #           270                                                               - Leu Lys Lys Leu Leu Gln Leu Asp Pro Val Le - #u Leu Leu Met Val Ser         #       285                                                                   - Ser Val Gln Lys Ser Trp Tyr Phe Pro Glu Il - #e Arg Met Val Asp Gly         #   300                                                                       - Ser Arg Glu Gln Leu His Lys Met Arg Val Gl - #u Leu Glu Thr Pro Gln         305                 3 - #10                 3 - #15                 3 -       #20                                                                           - Ala Leu Leu Ser Tyr Gly His Thr Leu Leu Se - #r Ile Phe Arg Ala Glu         #               335                                                           - Phe Ile Lys Gly Tyr Val Ser Lys Asn Ala Ly - #s Trp Pro Pro Val His         #           350                                                               - Leu Leu Pro Gly Cys Asp Lys Ser Ile Lys As - #n Ala Arg Glu Leu Gly         #       365                                                                   - Arg Trp Ser Pro Ala Phe Asp Arg Arg Trp Gl - #n Leu Phe Glu Lys Val         #   380                                                                       - Val Ile Leu Arg Ile Ala Asp Leu Asp Met As - #p Pro Asp Phe Asn Asp         385                 3 - #90                 3 - #95                 4 -       #00                                                                           - Ile Val Ser Asp Lys Ala Ile Ile Ser Ser Ar - #g Arg Asp Trp Val Phe         #               415                                                           - Glu Tyr Asn Ala Ala Ala Phe Trp Lys Lys Ty - #r Gly Glu Arg Leu Glu         #           430                                                               - Arg Pro Pro Ala Arg Ser Gly Pro Ser Arg Le - #u Val Asn Ala Leu Ile         #       445                                                                   - Asp Gly Arg Leu Asp Asn Ile Pro Ala Leu Le - #u Glu Pro Phe Tyr Arg         #   460                                                                       - Gly Ala Val Glu Phe Glu Asp Arg Leu Thr Va - #l Leu Val Pro Lys Glu         465                 4 - #70                 4 - #75                 4 -       #80                                                                           - Lys Glu Leu Lys Val Lys Gly Arg Phe Phe Se - #r Lys Gln Thr Leu Ala         #               495                                                           - Ile Arg Ile Tyr Gln Val Val Ala Glu Ala Al - #a Leu Lys Asn Glu Val         #           510                                                               - Met Pro Tyr Leu Lys Thr His Ser Met Thr Me - #t Ser Ser Thr Ala Leu         #       525                                                                   - Thr His Leu Leu Asn Arg Leu Ser His Thr Il - #e Thr Lys Gly Asp Ser         #   540                                                                       - Phe Val Ile Asn Leu Asp Tyr Ser Ser Trp Cy - #s Asn Gly Phe Arg Pro         545                 5 - #50                 5 - #55                 5 -       #60                                                                           - Glu Leu Gln Ala Pro Ile Cys Arg Gln Leu As - #p Gln Met Phe Asn Cys         #               575                                                           - Gly Tyr Phe Phe Arg Thr Gly Cys Thr Leu Pr - #o Cys Phe Thr Thr Phe         #           590                                                               - Ile Ile Gln Asp Arg Phe Asn Pro Pro Tyr Se - #r Leu Ser Gly Glu Pro         #       605                                                                   - Val Glu Asp Gly Val Thr Cys Ala Val Gly Th - #r Lys Thr Met Gly Glu         #   620                                                                       - Gly Met Arg Gln Lys Leu Trp Thr Ile Leu Th - #r Ser Cys Trp Glu Ile         625                 6 - #30                 6 - #35                 6 -       #40                                                                           - Ile Ala Leu Arg Glu Ile Asn Val Thr Phe As - #n Ile Leu Gly Gln Gly         #               655                                                           - Asp Asn Gln Thr Ile Ile Ile His Lys Ser Al - #a Ser Gln Asn Asn Gln         #           670                                                               - Leu Leu Ala Glu Arg Ala Leu Gly Ala Leu Ty - #r Lys His Ala Arg Leu         #       685                                                                   - Ala Gly His Asn Leu Lys Val Glu Glu Cys Tr - #p Val Ser Asp Cys Leu         #   700                                                                       - Tyr Glu Tyr Gly Lys Lys Leu Phe Phe Arg Gl - #y Val Pro Val Pro Gly         705                 7 - #10                 7 - #15                 7 -       #20                                                                           - Cys Leu Lys Gln Leu Ser Arg Val Thr Asp Se - #r Thr Gly Glu Leu Phe         #               735                                                           - Pro Asn Leu Tyr Ser Lys Leu Ala Cys Leu Th - #r Ser Ser Cys Leu Ser         #           750                                                               - Ala Ala Met Ala Asp Thr Ser Pro Trp Val Al - #a Leu Ala Thr Gly Val         #       765                                                                   - Cys Leu Tyr Leu Ile Glu Leu Tyr Val Glu Le - #u Pro Pro Ala Ile Met         #   780                                                                       - Gln Asp Glu Ser Leu Leu Thr Thr Leu Cys Le - #u Val Gly Pro Ser Ile         785                 7 - #90                 7 - #95                 8 -       #00                                                                           - Gly Gly Leu Pro Thr Pro Ala Thr Leu Pro Se - #r Val Phe Phe Arg Gly         #               815                                                           - Met Ser Asp Pro Leu Pro Phe Gln Leu Ala Le - #u Leu Gln Thr Leu Ile         #           830                                                               - Lys Thr Thr Gly Val Thr Cys Ser Leu Val As - #n Arg Val Val Lys Leu         #       845                                                                   - Arg Ile Ala Pro Tyr Pro Asp Trp Leu Ser Le - #u Val Thr Asp Pro Thr         #   860                                                                       - Ser Leu Asn Ile Ala Gln Val Tyr Arg Pro Gl - #u Arg Gln Ile Arg Arg         865                 8 - #70                 8 - #75                 8 -       #80                                                                           - Trp Ile Glu Glu Ala Ile Ala Thr Ser Ser Hi - #s Ser Ser Arg Ile Ala         #               895                                                           - Thr Phe Phe Gln Gln Pro Leu Thr Glu Met Al - #a Gln Leu Leu Ala Arg         #           910                                                               - Asp Leu Ser Thr Met Met Pro Leu Arg Pro Ar - #g Asp Met Ser Ala Leu         #       925                                                                   - Phe Ala Leu Ser Asn Val Ala Tyr Gly Leu Se - #r Ile Ile Asp Leu Phe         #   940                                                                       - Gln Lys Ser Ser Thr Val Val Ser Ala Ser Gl - #n Ala Val His Ile Glu         945                 9 - #50                 9 - #55                 9 -       #60                                                                           - Asp Val Ala Leu Glu Ser Val Arg Tyr Lys Gl - #u Ser Ile Ile Gln Gly         #               975                                                           - Leu Leu Asp Thr Thr Glu Gly Tyr Asn Met Gl - #n Pro Tyr Leu Glu Gly         #           990                                                               - Cys Thr Tyr Leu Ala Ala Lys Gln Leu Arg Ar - #g Leu Thr Trp Gly Arg         #      10050                                                                  - Asp Leu Val Gly Val Thr Met Pro Phe Val Al - #a Glu Gln Phe His Pro         #  10205                                                                      - His Ser Ser Val Gly Ala Lys Ala Glu Leu Ty - #r Leu Asp Ala Ile Ile         #               10401030 - #                1035                              - Tyr Cys Pro Gln Glu Thr Leu Arg Ser His Hi - #s Leu Thr Thr Arg Gly         #              10550                                                          - Asp Gln Pro Leu Tyr Leu Gly Ser Asn Thr Al - #a Val Lys Val Gln Arg         #          10705                                                              - Gly Glu Ile Thr Gly Leu Thr Lys Ser Arg Al - #a Ala Asn Leu Val Arg         #      10850                                                                  - Asp Thr Leu Val Leu His Gln Trp Tyr Lys Va - #l Arg Lys Val Thr Asp         #  11005                                                                      - Pro His Leu Asn Thr Leu Met Ala Arg Phe Le - #u Leu Glu Lys Gly Tyr         #               11201110 - #                1115                              - Thr Ser Asp Ala Arg Pro Ser Ile Gln Gly Gl - #y Thr Leu Thr His Arg         #              11350                                                          - Leu Pro Ser Arg Gly Asp Ser Arg Gln Gly Le - #u Thr Gly Tyr Val Asn         #          11505                                                              - Ile Leu Ser Thr Trp Leu Arg Phe Ser Ser As - #p Tyr Leu His Ser Phe         #      11650                                                                  - Ser Lys Ser Ser Asp Asp Tyr Thr Ile His Ph - #e Gln His Val Phe Thr         #  11805                                                                      - Tyr Gly Cys Leu Tyr Ala Asp Ser Val Ile Ar - #g Ser Gly Gly Val Ile         #               12001190 - #                1195                              - Ser Thr Pro Tyr Leu Leu Ser Ala Ser Cys Ly - #s Thr Cys Phe Glu Lys         #              12150                                                          - Ile Asp Ser Glu Glu Phe Val Leu Ala Cys Gl - #u Pro Gln Tyr Arg Gly         #          12305                                                              - Ala Glu Trp Leu Ile Ser Lys Pro Val Thr Va - #l Pro Glu Gln Ile Thr         #      12450                                                                  - Asp Ala Glu Val Glu Phe Asp Pro Cys Val Se - #r Ala Gly Tyr Cys Leu         #  12605                                                                      - Gly Ile Leu Ile Gly Lys Ser Phe Leu Val As - #p Ile Arg Ala Ser Gly         #               12801270 - #                1275                              - His Asp Ile Met Glu Gln Arg Thr Trp Ala As - #n Leu Glu Arg Phe Ser         #              12950                                                          - Val Ser Asp Met Gln Lys Leu Pro Trp Ser Il - #e Val Ile Arg Ser Leu         #          13105                                                              - Trp Arg Phe Leu Ile Gly Ala Arg Leu Leu Gl - #n Phe Glu Lys Ala Gly         #      13250                                                                  - Leu Ile Arg Met Leu Tyr Ala Ala Thr Gly Pr - #o Thr Pro Ser Phe Leu         #  13405                                                                      - Met Lys Val Phe Gln Asp Ser Ala Leu Leu Me - #t Asp Cys Ala Pro Leu         #               13601350 - #                1355                              - Asp Arg Leu Ser Pro Arg Ile Asn Phe His Se - #r Arg Gly Asp Leu Val         #              13750                                                          - Ala Lys Leu Val Leu Leu Pro Phe Ile Asn Pr - #o Gly Ile Val Glu Ile         #          13905                                                              - Glu Val Ser Gly Ile Asn Ser Lys Tyr His Al - #a Val Ser Glu Ala Asn         #      14050                                                                  - Met Asp Leu Tyr Ile Ala Ala Ala Lys Ser Va - #l Gly Val Lys Pro Thr         #  14205                                                                      - Gln Phe Val Glu Glu Thr Asn Asp Phe Thr Al - #a Arg Gly His His His         #               14401430 - #                1435                              - Gly Cys Tyr Ser Leu Ser Trp Ser Lys Ser Ar - #g Asn Gln Ser Gln Val         #              14550                                                          - Leu Lys Met Val Val Arg Lys Leu Lys Leu Cy - #s Val Leu Tyr Ile Tyr         #          14705                                                              - Pro Thr Val Asp Pro Ala Val Ala Leu Asp Le - #u Cys His Leu Pro Ala         #      14850                                                                  - Leu Thr Ile Ile Leu Val Leu Gly Gly Asp Pr - #o Ala Tyr Tyr Glu Arg         #  15005                                                                      - Leu Leu Glu Met Asp Leu Cys Gly Ala Val Se - #r Ser Arg Val Asp Ile         #               15201510 - #                1515                              - Pro His Ser Leu Ala Gly Arg Thr His Arg Gl - #y Phe Ala Val Gly Pro         #              15350                                                          - Asp Ala Gly Pro Gly Val Ile Arg Leu Asp Ar - #g Leu Glu Ser Val Cys         #          15505                                                              - Tyr Ala His Pro Cys Leu Glu Glu Leu Glu Ph - #e Asn Ala Tyr Leu Asp         #      15650                                                                  - Ser Glu Leu Val Asp Ile Ser Asp Met Cys Cy - #s Leu Pro Leu Ala Thr         #  15805                                                                      - Pro Cys Lys Ala Leu Phe Arg Pro Ile Tyr Ar - #g Ser Leu Gln Ser Phe         #               16001590 - #                1595                              - Arg Leu Ala Leu Met Asp Asn Tyr Ser Phe Va - #l Met Asp Leu Ile Met         #              16150                                                          - Ile Arg Gly Leu Asp Ile Arg Pro His Leu Gl - #u Glu Phe Asp Glu Leu         #          16305                                                              - Leu Val Val Gly Gln His Ile Leu Gly Gln Pr - #o Val Leu Val Glu Val         #      16450                                                                  - Val Tyr Tyr Val Gly Val Val Arg Lys Arg Pr - #o Val Leu Ala Arg His         #  16605                                                                      - Pro Trp Ser Ala Asp Leu Lys Arg Ile Thr Va - #l Gly Gly Arg Ala Pro         #               16801670 - #                1675                              - Cys Pro Ser Ala Ala Arg Leu Arg Asp Glu As - #p Cys Gln Gly Ser Leu         #              16950                                                          - Leu Val Gly Leu Pro Ala Gly Leu Thr Gln Le - #u Leu Ile Ile Asp             #          17105                                                              - (2) INFORMATION FOR SEQ ID NO:11:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 24 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA to genomic RN - #A                             -    (iii) HYPOTHETICAL:  NO                                                  -     (iv) ANTI-SENSE:  YES                                                   -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                #                24ACCC TGTA                                                  - (2) INFORMATION FOR SEQ ID NO:12:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 20 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA to genomic RN - #A                             -    (iii) HYPOTHETICAL: NO                                                   -     (iv) ANTI-SENSE: NO                                                     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                # 20               TGCA                                                       - (2) INFORMATION FOR SEQ ID NO:13:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 22 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA to genomic RN - #A                             -    (iii) HYPOTHETICAL: NO                                                   -     (iv) ANTI-SENSE: YES                                                    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                                #                 22GAA GC                                                    - (2) INFORMATION FOR SEQ ID NO:14:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 20 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA to genomic RN - #A                             -    (iii) HYPOTHETICAL: NO                                                   -     (iv) ANTI-SENSE: YES                                                    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                                # 20               GCTG                                                       - (2) INFORMATION FOR SEQ ID NO:15:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 30 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA to mRNA                                        -    (iii) HYPOTHETICAL: NO                                                   -     (iv) ANTI-SENSE: NO                                                     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                                #           30     AAAA CATTCCTATC                                            - (2) INFORMATION FOR SEQ ID NO:16:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 21 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA to mRNA                                        -    (iii) HYPOTHETICAL: NO                                                   -     (iv) ANTI-SENSE: YES                                                    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                                #21                CGAA T                                                     - (2) INFORMATION FOR SEQ ID NO:17:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 19 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA to mRNA                                        -    (iii) HYPOTHETICAL: NO                                                   -     (iv) ANTI-SENSE: NO                                                     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                                # 19               TTT                                                        - (2) INFORMATION FOR SEQ ID NO:18:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 25 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA to mRNA                                        -    (iii) HYPOTHETICAL: NO                                                   -     (iv) ANTI-SENSE: NO                                                     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:                                #               25 GCGA CCATC                                                 - (2) INFORMATION FOR SEQ ID NO:19:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 8910 base                                                         (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA to genomic RN - #A                             -    (iii) HYPOTHETICAL: NO                                                   -     (iv) ANTI-SENSE: NO                                                     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:                                - GTTGCGTTAA CAACAAACCA CTCATCATTC TTCTAACAAA ATGAACACAC GC - #AATGCCAC         60                                                                          - CCAAGAGACG CCTGGTTGAT GACGCCGATG CCATGGAGGA TCAAGATCTA TA - #TGAACCCC        120                                                                          - CAGCGAGCCT CCCTAAGCTC CCTGGGAAAT TCCTACAATA CACCGTTGGG GG - #GTCTGACC        180                                                                          - CGCATCCGGG TATAGGGCAT GAGAAAGACA TCAGGCAGAA CGCAGTGGCA TT - #GTTAGACC        240                                                                          - AGTCACGGCG CGATATGTTT CACACAGTAA CGCCTAGCCT TGTGTTTCTA TG - #TTTGCTAA        300                                                                          - TCCCAGGACT GCACGCTGCG TTTGTTCACG GAGGGGTGCC TCGTGAATCC TA - #CCTGTCGA        360                                                                          - CGCCTGTCAC GCGTGGAGAA CAGACTGTTG TTAAGACTGC GAAGTTTTAC GG - #GGAAAAGA        420                                                                          - CGACGCAGCG TGATCTCACC GAGCTGGAGA TCTCCTCTAT CTTCAGCCAT TG - #TTGCTCAT        480                                                                          - TACTAATAGG GGTTGTGATA GGATCGTCGT CTAAGATCAA AGCAGGAGCC GA - #GCAGATCA        540                                                                          - AGAAAAGGTT TAAAACTATG ATGGCAGCCT TAAACCGGCC ATCCCATGGT GA - #GACTGCTA        600                                                                          - CACTACTCCA GATGTTTAAT CCACATGAGG CTATAGATTG GATTAACGGC CA - #ACCCTGGG        660                                                                          - TAGGCTCCTT TGTGTTGTCT CTACTAACTA CAGACTTTGA GTCCCCAGGT AA - #AGAATTTA        720                                                                          - TGGACCAGAT TAAGCTTGTC GCAAGTTATG CACAGATGAC TACGTACACT AC - #TATAAAGG        780                                                                          - AGTACCTCGC AGAATGCATG GATGCTACCC TTACAATCCC CGTAGTTGCA TA - #TGAGATCC        840                                                                          - GTGACTTTTT AGAAGTTTCA GCAAAGCTTA AGGAGGATCA TGCTGACCTG TT - #CCCGTTTC        900                                                                          - TGGGGGCCAT TAGACACCCC GACGCTATCA AGCTGGCGCC ACGAAGCTTT CC - #CAATCTGG        960                                                                          - CCTCCGCAGC GTTTTACTGG AGTAAGAAGG AAAACCCCAC AATGGCAGGC TA - #CCGGGCCT       1020                                                                          - CCACCATCCA GCCGGGCGCA AGTGTCAAGG AAACCCAGCT TGCCCGGTAT AG - #GCGCCGCG       1080                                                                          - AGATATCTCG TGGAGAGGAC GGGGCAGAGC TCTCAGGTGA GATCTCTGCC AT - #AATGAAGA       1140                                                                          - TGATAGGTGT GACTGGTCTA AACTAAAAAA CAATGAACAA ACCAATAAAA AA - #CCAAATGC       1200                                                                          - GGCAAACCCT CCGCGACCTG CGATGAGCTC CGACCTCCGG CTGACATTGC TT - #GAACTAGT       1260                                                                          - CAGGAGGCTC AATGGCAACG CGACCATCGA GTCTGGTCGA CTCCCTGGAG GA - #CGAAGAAG       1320                                                                          - ATCCCCAGAC ACTACGACGG GAACGACCGG GGTCACCAAG ACCACGGAAG GT - #CCCAAGGA       1380                                                                          - ATGCATTGAC CCAACCAGTA GACCAGCTCC TGAAGGACCT CAGGAAGAAC CC - #CTCCATGA       1440                                                                          - TCTCAGACCC AGACCAGCGA ACCGGAAGGG AGCAGCTGTC GAATGATGAG CT - #AATCAAGA       1500                                                                          - AGTTAGTGAC GGAGCTGGCC GAGAATAGCA TGATCGAGGC TGAGGAGGTG CG - #GGGCACTC       1560                                                                          - TTGGAGACAT CTCGGCTCGT ATCGAGGCAG GGTTTGAGTC CCTGTCCGCC CT - #CCAAGTGG       1620                                                                          - AAACCATCCA GACAGCTCAG CGGTGCGATC ACTCCGACAG CATCAGGATC CT - #CGGCGAGA       1680                                                                          - ACATCAAGAT ACTAGATCGC TCCATGAAGA CAATGATGGA GACAATGAAG CT - #CATGATGG       1740                                                                          - AGAAGGTGGA TCTCCTCTAC GCATCAACCG CCGTTGGGAC CTCTGCACCC AT - #GTTGCCCT       1800                                                                          - CCCATCCTGC ACCTCCGCGC ATTTATCCCC AGCTCCCAAG TGCCCCGACA AC - #GGATGAAT       1860                                                                          - GGGACATCAT ACCATAAAAA AATCGAATCA CCATGAATTC AAAACATTCC TA - #TGTGGAGC       1920                                                                          - TCAAGGACAA GGTAATCGTC CCTGGATGGC CCACACTGAT GCTTGAGATA GA - #CTTTGTAG       1980                                                                          - GGGGGACTTC ACGGAACCAG TTCCTTAACA TCCCATTTCT TTCAGTGAAA GA - #GCCTCTGC       2040                                                                          - AGCTTCCACG CGAGAAGAAG TTGACCGACT ACTTTACTAT TGACGTAGAA CC - #AGCAGGTC       2100                                                                          - ATTCCCTGGT CAATATATAC TTCCAGATTG ACGACTTCTT GCTCCTAACA CT - #CAACTCAC       2160                                                                          - TATCTGTGTA CAAGGACCCG ATTAGAAAAT ACATGTTCCT ACGCCTCAAC AA - #GGACCAGA       2220                                                                          - GCAAGCACGC AATCAATGCA GCCTTCAATG TCTTTTCTTA TCGGCTTCGG AA - #CATTGGTG       2280                                                                          - TTGGTCCTCT CGGCCCGGAC ATTCGATCTT CAGGGCCTTA GCTGCAATAC TG - #ACTCCACT       2340                                                                          - CCTGGACTGA TTGACCTGGA GATAAGGCGA CTTTGCCACA CCCCAACGGA AA - #ATGTCATT       2400                                                                          - TCATGCGAGG TTAGTTATCT CAACCACACG ACTATTAGCC TCCCGGCAGT CC - #ACACATCA       2460                                                                          - TGCCTCAAGT ACCACTGCAA AACCTATTGG GGATTCTTTG GTAGCTACAG CG - #CTGACCGA       2520                                                                          - ATCATAAATC GGTACACTGG TACTGTTAAG GGTTGTCTAA ACAACTCAGC AC - #CAGAGGAC       2580                                                                          - CCCTTCGAGT GCAACTGGTT CTACTGCTGC TCGGCGATTA CAACAGAGAT CT - #GCCGATGC       2640                                                                          - TCTATTACAA ATGTCACGGT GGCTGTGCAA ACATTCCCAC CGTTCATGTA CT - #GCAGTTTT       2700                                                                          - GCAGACTGCA GTACCGTGAG CCAACAGGAG CTAGAGAGTG GAAAGGCAAT GC - #TGAGCGAT       2760                                                                          - GGCAGTACAT TAACTTATAC CCCGTATATC CTACAGTCAG AAGTCGTGAA CA - #AAACCCTC       2820                                                                          - AATGGGACCA TACTCTGCAA CTCATCCTCT AAGATAGTTT CCTTCGATGA AT - #TTAGGCGT       2880                                                                          - TCATACTCCC TAACGAATGG TAGTTACCAG AGCTCATCAA TCAATGTGAC GT - #GTGCAAAC       2940                                                                          - TACACGTCGT CCTGCCGGCC CAGGTTGAAA AGGCGGCGTA GGGACACCCA GC - #AGATTGAG       3000                                                                          - TATCTAGTTC ACAAGCTTAG GCCCACACTG AAAGATGCAT GGGAGGACTG TG - #AGATCCTC       3060                                                                          - CAGTCTCTGC TCCTAGGGGT GTTTGGTACT GGGATCGCAA GTGCTTCTCA AT - #TTTTGAGG       3120                                                                          - AGCTGGCTCA ACCACCCTGA CATCATCGGG TATATAGTTA ATGGAGTTGG GG - #TTGTCTGG       3180                                                                          - CAATGCCATC GTGTTAATGT CACGTTCATG GCGTGGAATG AGTCCACCTA TT - #ACCCTCCA       3240                                                                          - GTAGATTACA ATGGGCGGAA GTACTTCCTG AATGATGAGG GAAGGTTACA AA - #CAAACACC       3300                                                                          - CCCGAGGCAA GGCCAGGGCT TAAGCGGGTC ATGTGGTTCG GCAGGTACTT CC - #TAGGGACA       3360                                                                          - GTAGGGTCTG GGGTGAAACC GAGGAGGATT CGGTACAATA AGACCTCACA TG - #ACTACCAC       3420                                                                          - CTGGAGGAGT TTGAGGCAAG TCTCAACATG ACCCCTCAGA CCAGTATCGC CT - #CGGGTCAT       3480                                                                          - GAGACAGACC CCATAAATCA TGCCTACGGA ACGCAGGCTG ATCTCCTTCC AT - #ACACCAGG       3540                                                                          - TCTAGTAATA TAACATCTAC GGATACAGGC TCAGGCTGGG TGCACATCGG CC - #TACCCTCA       3600                                                                          - TTTGCTTTCC TCAATCCCCT CGGGTGGCTC AGGGACCTAC TTGCATGGGC AG - #CCTGGTTG       3660                                                                          - GGTGGGGTTC TATACTTAAT AAGTCTTTGT GTTTCCTTAC CAGCCTCCTT CG - #CGAGGAGG       3720                                                                          - AGACGCCTCG GCCGGTGGCA GGAATAAACC GTACCGACCA GTCTCTTAAA AA - #CCCTCTCC       3780                                                                          - TCGGAACAGA GGTCTCTTTC TGCCTTAAGT CGAGCTCACT CCCCCATCAT GT - #ACGAGCAC       3840                                                                          - TAGGCCAGAT TAAAGCAAGG AACCTGGCAT CCTGTGACTA TTACTTGCTA TT - #CCGCCAAG       3900                                                                          - TTGTATTGCC CCCTGAAGTA TATCCCATTG GTGTTCTAAT AAGAGCTGCG GA - #GGCTATAC       3960                                                                          - TAACAGTTAT AGTATCAGCT TGGAAGCTGG ATCATATGAC GAAGACCCTA TA - #CTCCTCTG       4020                                                                          - TGAGATATGC ACTCACCAAT CCCCGGGTCC GAGCCCAACT TGAGCTTCAC AT - #TGCCTACC       4080                                                                          - AGCGCATAGT GGGTCAGGTC TCGTACAGCC GGGAGGCAGA CATAGGGCCA AA - #AAGGCTTG       4140                                                                          - GGAATATGTC ATTGCAATTC ATCCAATCTC TCGTTATTGC CACCATAGAC AC - #GACAAGCT       4200                                                                          - GCCTAATGAC CTACAACCAC TTTCTTGCTG CAGCAGACAC AGCCAAGAGC AG - #ATGCCATC       4260                                                                          - TCCTAATCGC CTCAGTGGTC CAGGGGGCCC TTTGGGAACA AGGGTCATTT CT - #TGATCATA       4320                                                                          - TAATCAACAT GATCGACATA ATTGACTCAA TCAACCTCCC CCATGATGAT TA - #CTTCACAA       4380                                                                          - TTATTAAGTC TATCTTTCCC TACTCCCAAG GGCTTGTTAT GGGGAGGCAT AA - #TGTATCAG       4440                                                                          - TCTCCTCTGA TTTCGCGTCC GTATTTGCCA TTCCTGAATT ATGCCCGCAA CT - #AGACAGCT       4500                                                                          - TACTAAAAAA ACTGCTCCAA CTTGACCCCG TTCTCCTCCT CATGGTCTCT TC - #GGTGCAGA       4560                                                                          - AGTCATGGTA CTTCCCTGAG ATCCGAATGG TCGACGGGTC ACGGGAGCAG CT - #CCACAAGA       4620                                                                          - TGCGTGTCGA GCTGGAAACG CCCCAAGCCC TGCTGTCGTA CGGCCATACC CT - #CCTGTCAA       4680                                                                          - TATTTCGGGC AGAGTTTATC AAAGGCTATG TCTCAAAGAA TGCGAAGTGG CC - #GCCCGTAC       4740                                                                          - ACCTGCTCCC AGGCTGTGAC AAATCCATAA AAAATGCGAG AGAGCTGGGC CG - #CTGGAGCC       4800                                                                          - CGGCATTTGA CCGACGATGG CAGCTCTTCG AGAAGGTTGT CATTCTAAGA AT - #TGCTGACC       4860                                                                          - TAGATATGGA TCCCGACTTC AACGATATTG TTAGCGATAA GGCGATAATC AG - #CTCAAGAA       4920                                                                          - GGGACTGGGT ATTCGAGTAC AATGCAGCGG CCTTTTGGAA GAAATACGGT GA - #ACGGTTGG       4980                                                                          - AGAGGCCTCC TGCCAGGTCG GGACCGTCAC GACTTGTGAA TGCTCTAATC GA - #TGGACGCT       5040                                                                          - TAGACAATAT CCCAGCCCTG CTAGAGCCAT TTTACAGGGG AGCGGTTGAG TT - #CGAGGATC       5100                                                                          - GGTTGACTGT GCTCGTGCCT AAGGAGAAAG AGTTAAAGGT AAAGGGAAGG TT - #CTTCTCGA       5160                                                                          - AGCAAACATT GGCAATCAGG ATATATCAGG TTGTTGCTGA AGCTGCACTT AA - #GAATGAGG       5220                                                                          - TTATGCCATA CCTAAAGACA CACTCAATGA CCATGAGCTC AACGGCTCTA AC - #TCACCTTC       5280                                                                          - TTAACCGGCT ATCACATACT ATCACTAAGG GTGACTCCTT TGTTATTAAC CT - #TGACTATA       5340                                                                          - GTTCCTGGTG CAACGGTTTC CGACCAGAAC TGCAGGCCCC AATCTGTCGT CA - #GTTGGATC       5400                                                                          - AGATGTTCAA TTGCGGGTAC TTCTTCAGGA CTGGGTGCAC ACTGCCATGC TT - #TACCACGT       5460                                                                          - TTATTATTCA AGACAGGTTC AACCCGCCCT ATTCCCTCAG TGGTGAGCCC GT - #TGAAGACT       5520                                                                          - GAGTTACATG CGCGGTTGGG ACTAAAACAA TGGGGGAGGG CATGAGGCAG AA - #ACTATGGA       5580                                                                          - CAATCCTTAC GAGCTGCTGG GAGATAATTG CTCTTCGGGA AATTAACGTG AC - #GTTTAACA       5640                                                                          - TACTAGGCCA AGGTGATAAT CAGACAATCA TCATACATAA ATCTGCAAGC CA - #AAATAACC       5700                                                                          - AGCTATTAGC GGAGCGAGCA CTAGGGGCCC TGTACAAGCA TGCTAGATTA GC - #TGGCCATA       5760                                                                          - ACCTCAAGGT AGAGGAATGC TGGGTGTCAG ATTGTCTGTA TGAGTATGGA AA - #GAAGCTTT       5820                                                                          - TCTTCCGTGG TGTACCTGTC CCGGGCTGTT TGAAGCAGCT CTCACGGGTG AC - #GGATTCTA       5880                                                                          - CTGGAGAGCT ATTCCCAAAC CTATACTCAA AGTTAGCCTG CTTAACATCA TC - #GTGTTTAA       5940                                                                          - GCGCAGCGAT GGCAGACACA TCTCCATGGG TGGCACTCGC GACAGGTGTC TG - #TCTGTATC       6000                                                                          - TTATCGAGTT ATATGTTGAG CTGCCTCCAG CAATCATGCA GGATGAGTCG CT - #ATTGACGA       6060                                                                          - CCCTCTGCCT CGTAGGCCCA TCCATTGGTG GGCTTCCGAC CCCTGCAACC CT - #ACCCAGTG       6120                                                                          - TCTTTTTCAG AGGAATGTCC GACCCACTGC CCTTTCAGCT AGCACTCTTG CA - #GACCCTCA       6180                                                                          - TTAAGACGAC AGGGGTGACC TGTAGCTTGG TGAATCGTGT GGTCAAGTTA CG - #GATAGCAC       6240                                                                          - CCTATCCAGA CTGGCTCTCT CTAGTGACTG ACCCGACCTC ACTCAACATT GC - #CCAAGTGT       6300                                                                          - ACCGGCCAGA ACGTCAGATC AGGAGGTGGA TTGAGGAAGC GATAGCGACA AG - #CTCACACT       6360                                                                          - CGTCACGCAT AGCAACTTTC TTCCAGCAGC CCCTCACGGA GATGGCTCAG TT - #GCTTGCGA       6420                                                                          - GGGACCTTTC AACAATGATG CCTCTTCGAC CCCGGGATAT GTCGGCCTTA TT - #CGCATTAT       6480                                                                          - CAAATGTCGC ATACGGTTTA AGCATTATAG ATCTATTTCA AAAATCCTCT AC - #CGTTGTTT       6540                                                                          - CTGCAAGTCA AGCTGTCCAT ATCGAGGATG TTGCCCTAGA GAGTGTAAGG TA - #TAAGGAAT       6600                                                                          - CTATCATCCA GGGTCTGTTA GACACCACTG AGGGGTATAA CATGCAACCT TA - #TTTGGAAG       6660                                                                          - GTTGCACTTA CCTTGCAGCC AAACAGTTAC GTAGGTTGAC ATGGGGTCGA GA - #CCTAGTTG       6720                                                                          - GAGTCACAAT GCCGTTTGTT GCCGAGCAAT TCCATCCTCA CAGTTCTGTG GG - #TGCAAAGG       6780                                                                          - CGGAACTCTA CCTCGACGCT ATTATATACT GCCCACAGGA GACATTGCGG TC - #ACACCATC       6840                                                                          - TGACTACCAG GGGGGACCAG CCGCTTTACC TCGGATCCAA TACGGCTGTC AA - #GGTCCAGC       6900                                                                          - GAGGTGAGAT CACGGGCCTA ACAAAGTCAA GGGCTGCAAA TCTAGTCAGG GA - #CACTCTCG       6960                                                                          - TTCTCCATCA GTGGTATAAA GTCCGTAAAG TTACCGATCC ACACTTGAAC AC - #CCTCATGG       7020                                                                          - CACGCTTCTT ACTTGAGAAG GGGTACACAT CTGACGCTCG ACCTAGCATC CA - #GGGTGGGA       7080                                                                          - CCCTCACGCA TCGTCTCCCA TCCCGCGGAG ACTCACGGCA GGGGCTTACT GG - #GTATGTAA       7140                                                                          - ATATACTAAG TACGTGGCTT CGATTCTCAA GTGATTATCT TCACTCTTTC TC - #GAAATCAT       7200                                                                          - CAGACGACTA TACAATCCAC TTTCAGCATG TATTCACATA CGGTTGCCTC TA - #TGCTGATT       7260                                                                          - CGGTGATTAG ATCGGGCGGT GTTATTTCCA CTCCTTACCT TTTGAGTGCA AG - #TTGTAAAA       7320                                                                          - CATGCTTTGA GAAGATAGAC TCAGAGGAGT TCGTCCTGGC ATGTGAACCC CA - #ATACAGGG       7380                                                                          - GTGCTGAGTG GCTGATATCA AAGCCAGTCA CTGTCCCTGA GCAGATAACT GA - #TGCTGAAG       7440                                                                          - TCGAGTTTGA CCCCTGTGTG AGTGCGGGTT ATTGTCTCGG GATTCTCATT GG - #CAAGTCAT       7500                                                                          - TCTTAGTTGA CATAAGGGCA AGTGGGCATG ATATCATGGA GCAGCGGACA TG - #GGCTAACC       7560                                                                          - TGGAGAGGTT TTCTGTATCG GACATGCAGA AACTTCCGTG GAGTATTGTA AT - #TCGGTCTC       7620                                                                          - TCTGGAGATT CCTTATTGGC GCACGGCTCC TTCAGTTTGA GAAGGCTGGC CT - #CATTAGAA       7680                                                                          - TGCTGTATGC TGCGACAGGT CCAACCCCTA GCTTCCTAAT GAAAGTTTTT CA - #AGACTCAG       7740                                                                          - CCCTCCTCAT GGACTGCGCA CCCCTCGATC GGCTGTCCCC TAGGATCAAC TT - #TCATAGTC       7800                                                                          - GGGGAGACCT CGTTGCTAAG CTTGTTTTAT TGCCCTTCAT CAACCCGGGT AT - #AGTGGAGA       7860                                                                          - TTGAAGTGTC TGGAATTAAT AGCAAGTACC ATGCAGTATC GGAGGCCAAT AT - #GGATCTGT       7920                                                                          - ACATCGCTGC TGCCAAGTCT GTGGGCGTGA AGCCCACACA GTTTGTTGAG GA - #AACAAACG       7980                                                                          - ACTTTACGGC CCGCGGCCAC CACCATGGTT GTTATTCCCT TTCTTGGTCT AA - #GTCACGCA       8040                                                                          - ATCAATCACA GGTCCTAAAG ATGGTAGTAC GGAAGCTGAA GCTCTGTGTC CT - #GTATATAT       8100                                                                          - ACCCCACAGT CGATCCCGCC GTTGCTCTCG ACCTGTGCCA TCTACCAGCA TT - #AACTATAA       8160                                                                          - TCCTAGTGCT CGGCGGTGAC CCAGCGTACT ATGAGCGATT ACTTGAGATG GA - #CCTGTGCG       8220                                                                          - GGGCTGTGTC AAGTCGAGTC GATATCCCCC ATTCTCTGGC TGGCAGAACG CA - #CAGGGGGT       8280                                                                          - TCGCAGTGGG CCCAGACGCT GGTCCAGGTG TAATTAGACT CGACAGGTTA GA - #GTCAGTTT       8340                                                                          - GTTATGCTCA CCCCTGTTTA GAGGAACTAG AGTTTAATGC ATATCTAGAC TC - #TGAGTTGG       8400                                                                          - TTGACATTAG TGATATGTGC TGCCTCCCCT TAGCGACACC CTGTAAGGCC CT - #TTTCAGGC       8460                                                                          - CAATATATCG GAGCTTACAG TCGTTCAGGT TAGCCTTAAT GGACAACTAT AG - #TTTTGTCA       8520                                                                          - TGGACCTCAT TATGATCCGA GGACTGGACA TTAGGCCTCA CCTTGAGGAA TT - #TGACGAGC       8580                                                                          - TGCTTGTGGT AGGACAGCAC ATCCTCGGCC AGCCCGTCCT AGTAGAGGTT GT - #TTACTACG       8640                                                                          - TTGGAGTTGT TAGGAAGCGC CCTGTGTTAG CGAGGCATCC GTGGTCAGCA GA - #TCTTAAGC       8700                                                                          - GAATTACTGT GGGGGGGCGG GCTCCCTGCC CCTCTGCTGC CAGATTGCGT GA - #TGAGGATT       8760                                                                          - GTCAGGGGTC TCTGTTGGTT GGGCTTCCTG CTGGGTTGAC GCAGTTATTG AT - #AATTGATT       8820                                                                          - AAGATCAAGC CACCTACTAC CCTATTCTTA AAAAACCATA TGTCAGTGGT GC - #AGTGCTTG       8880                                                                          #         8910     TGTT GTAGCGCGTT                                            - (2) INFORMATION FOR SEQ ID NO:20:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 8 amino                                                           (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -    (iii) HYPOTHETICAL:   NO                                                 -     (iv) ANTI-SENSE:  NO                                                    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:                                - Met Ala Thr Arg Pro Ser Ser Leu                                             1               5                                                             - (2) INFORMATION FOR SEQ ID NO:21:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 12 amino                                                          (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -    (iii) HYPOTHETICAL:   NO                                                 -     (iv) ANTI-SENSE:  NO                                                    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:                                - Asn Ala Leu Thr Gln Pro Val Asp Gln Leu Le - #u Lys                         #                10                                                           - (2) INFORMATION FOR SEQ ID NO:22:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 8 amino                                                           (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -    (iii) HYPOTHETICAL:   NO                                                 -     (iv) ANTI-SENSE:  NO                                                    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:                                - Asp Gln Pro Thr Gly Arg Glu Gln                                             1               5                                                             - (2) INFORMATION FOR SEQ ID NO:23:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 8 amino                                                           (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -    (iii) HYPOTHETICAL:   NO                                                 -     (iv) ANTI-SENSE:  NO                                                    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:                                - Val Arg Gly Thr Leu Gly Asp Ile                                             1               5                                                             - (2) INFORMATION FOR SEQ ID NO:24:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 8 amino                                                           (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -    (iii) HYPOTHETICAL:   NO                                                 -     (iv) ANTI-SENSE:  NO                                                    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:                                - Thr Ala Gln Arg Cys Asp His Ser                                             1               5                                                             - (2) INFORMATION FOR SEQ ID NO:25:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 12 amino                                                          (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -    (iii) HYPOTHETICAL:   NO                                                 -     (iv) ANTI-SENSE:  NO                                                    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:                                - Met Glu Thr Met Lys Leu Met Met Glu Lys Va - #l Asp                         #                10                                                           - (2) INFORMATION FOR SEQ ID NO:26:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 8 amino                                                           (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -    (iii) HYPOTHETICAL:   NO                                                 -     (iv) ANTI-SENSE:  NO                                                    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:                                - Pro Met Leu Pro Ser His Pro Ala                                             1               5                                                             - (2) INFORMATION FOR SEQ ID NO:27:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 8 amino                                                           (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -    (iii) HYPOTHETICAL:   NO                                                 -     (iv) ANTI-SENSE:  NO                                                    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:                                - Thr Ala Asp Glu Trp Asp Ile Ile                                             1               5                                                             - (2) INFORMATION FOR SEQ ID NO:28:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 8 amino                                                           (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -    (iii) HYPOTHETICAL:   NO                                                 -     (iv) ANTI-SENSE:  NO                                                    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:                                - Met Asn Ser Lys His Ser Tyr Val                                             1               5                                                             - (2) INFORMATION FOR SEQ ID NO:29:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 8 amino                                                           (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -    (iii) HYPOTHETICAL:   NO                                                 -     (iv) ANTI-SENSE:  NO                                                    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:                                - Thr Leu Met Leu Glu Ile Asp Phe                                             1               5                                                             - (2) INFORMATION FOR SEQ ID NO:30:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 12 amino                                                          (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -    (iii) HYPOTHETICAL:   NO                                                 -     (iv) ANTI-SENSE:  NO                                                    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:                                - Gly His Ser Leu Val Asn Ile Tyr Phe Gln Il - #e Asp                         #                10                                                           - (2) INFORMATION FOR SEQ ID NO:31:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 8 amino                                                           (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -    (iii) HYPOTHETICAL:   NO                                                 -     (iv) ANTI-SENSE:  NO                                                    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:                                - Tyr Lys Asp Pro Ile Arg Lys Tyr                                             1               5                                                             - (2) INFORMATION FOR SEQ ID NO:32:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 8 amino                                                           (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -    (iii) HYPOTHETICAL:   NO                                                 -     (iv) ANTI-SENSE:  NO                                                    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:                                - Ala Phe Asn Val Phe Ser Tyr Arg                                             1               5                                                             - (2) INFORMATION FOR SEQ ID NO:33:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 484 amino                                                         (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -    (iii) HYPOTHETICAL:   NO                                                 -     (iv) ANTI-SENSE:  NO                                                    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33:                                - Thr Trp Pro Pro Lys His Ile Val Asp Leu Va - #l Gly Asp Thr Trp             #                 15                                                          - His Lys Leu Pro Ile Thr Gln Ile Phe Glu Il - #e Pro Glu Ser Met             #                30                                                           - Asp Pro Ser Glu Ile Leu Asp Asp Lys Ser Hi - #s Ser Phe Thr Arg             #                45                                                           - Thr Arg Leu Ala Ser Trp Leu Ser Glu Asn Ar - #g Gly Gly Pro Val             #                60                                                           - Pro Ser Glu Lys Val Ile Ile Thr Ala Leu Se - #r Lys Pro Pro Val             #                75                                                           - Asn Pro Arg Glu Phe Leu Arg Ser Ile Asp Le - #u Gly Gly Leu Pro             #                90                                                           - Asp Glu Asp Leu Ile Ile Gly Leu Lys Pro Ly - #s Glu Arg Glu Leu             #                105                                                          - Lys Ile Glu Gly Arg Phe Phe Ala Leu Met Se - #r Trp Asn Leu Arg             #               120                                                           - Leu Tyr Phe Val Ile Thr Glu Lys Leu Leu Al - #a Asn Tyr Ile Leu             #               135                                                           - Pro Leu Phe Asp Ala Leu Thr Met Thr Asp As - #n Leu Asn Lys Val             #               150                                                           - Phe Lys Lys Leu Ile Asp Arg Val Thr Gly Gl - #n Gly Leu Leu Asp             #               165                                                           - Tyr Ser Arg Val Thr Tyr Ala Phe His Leu As - #p Tyr Glu Lys Trp             #               180                                                           - Asn Asn His Gln Arg Leu Glu Ser Thr Glu As - #p Val Phe Ser Val             #               195                                                           - Leu Asp Gln Val Phe Gly Leu Lys Arg Val Ph - #e Ser Arg Thr His             #               210                                                           - Glu Phe Phe Gln Lys Ala Trp Ile Tyr Tyr Se - #r Asp Arg Ser Asp             #               225                                                           - Leu Ile Gly Leu Arg Glu Asp Gln Ile Tyr Cy - #s Leu Asp Ala Ser             #               240                                                           - Asn Gly Pro Thr Cys Trp Asn Gly Gln Asp Gl - #y Gly Leu Glu Gly             #               255                                                           - Leu Arg Gln Lys Gly Trp Ser Leu Val Ser Le - #u Leu Met Ile Asp             #               270                                                           - Arg Glu Ser Gln Ile Arg Asn Thr Arg Thr Ly - #s Ile Leu Ala Gln             #               285                                                           - Gly Asp Asn Gln Val Leu Cys Pro Thr Tyr Me - #t Leu Ser Pro Gly             #               300                                                           - Leu Ser Gln Glu Gly Leu Leu Tyr Glu Leu Gl - #u Arg Ile Ser Arg             #               315                                                           - Asn Ala Leu Ser Ile Tyr Arg Ala Val Glu Gl - #u Gly Ala Ser Lys             #               330                                                           - Leu Gly Leu Ile Ile Lys Lys Glu Glu Thr Me - #t Cys Ser Tyr Asp             #               345                                                           - Phe Leu Ile Tyr Gly Lys Thr Pro Leu Phe Ar - #g Gly Asn Ile Leu             #               360                                                           - Val Pro Glu Ser Lys Arg Trp Ala Arg Val Se - #r Cys Val Ser Asn             #               375                                                           - Asp Gln Ile Val Asn Leu Ala Asn Ile Met Se - #r Thr Val Ser Thr             #               390                                                           - Asn Ala Leu Thr Val Ala Gln His Ser Gln Se - #r Leu Ile Lys Pro             #               405                                                           - Met Arg Asp Phe Leu Leu Met Ser Val Gln Al - #a Val Phe His Tyr             #               420                                                           - Leu Leu Phe Ser Pro Ile Leu Lys Gly Arg Va - #l Tyr Lys Ile Leu             #               435                                                           - Ser Ala Glu Gly Glu Ser Phe Leu Leu Ala Me - #t Ser Arg Ile Ile             #               450                                                           - Tyr Leu Asp Pro Ser Leu Gly Gly Ile Ser Gl - #y Met Ser Leu Gly             #               465                                                           - Arg Phe His Ile Arg Gln Phe Ser Asp Pro Va - #l Ser Glu Gly Leu             #               480                                                           - Ser Phe Trp Arg                                                                         484                                                               - (2) INFORMATION FOR SEQ ID NO:34:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 483 amino                                                         (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -    (iii) HYPOTHETICAL:   NO                                                 -     (iv) ANTI-SENSE:  NO                                                    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:                                - Thr Trp Pro Thr Ala Ala Lys Ile Gln Asp Ph - #e Gly Asp Asn Trp             #                 15                                                          - His Lys Leu Pro Leu Ile Gln Cys Phe Glu Il - #e Pro Asp Leu Ile             #                30                                                           - Asp Pro Ser Val Ile Tyr Ser Asp Lys Ser Hi - #s Ser Met Asn Lys             #                45                                                           - Lys Glu Val Ile Gln His Val Arg Ser Lys Pr - #o Asn Ile Pro Ile             #                60                                                           - Pro Ser Asn Lys Val Leu Gln Thr Met Leu Th - #r Asn Arg Ala Thr             #                75                                                           - Asn Trp Lys Ala Phe Leu Lys Asp Ile Asp Gl - #u Asn Gly Leu Asp             #                90                                                           - Asp Asp Asp Leu Ile Ile Gly Leu Lys Gly Ly - #s Glu Arg Glu Leu             #                105                                                          - Lys Ile Ala Gly Arg Phe Phe Ser Leu Met Se - #r Trp Arg Leu Arg             #               120                                                           - Glu Tyr Phe Val Ile Thr Glu Tyr Leu Ile Ly - #s Thr Tyr Tyr Val             #               135                                                           - Pro Leu Phe Lys Gly Leu Thr Met Ala Asp As - #p Leu Thr Ser Val             #               150                                                           - Ile Lys Lys Met Met Asp Ser Ser Ser Gly Gl - #n Gly Leu Asp Asp             #               165                                                           - Tyr Ser Ser Val Cys Leu Ala Asn His Ile As - #p Tyr Glu Lys Trp             #               180                                                           - Asn Asn His Gln Arg Lys Glu Ser Asn Gly Pr - #o Ile Phe Arg Val             #               195                                                           - Met Gly Gln Phe Leu Gly Tyr Pro Ser Leu Il - #e Glu Arg Thr His             #               210                                                           - Glu Phe Phe Glu Lys Ser Leu Ile Tyr Tyr As - #n Gly Arg Pro Asp             #               225                                                           - Leu Met Thr Ile Arg Asn Gly Thr Leu Cys As - #n Ser Thr Lys His             #               240                                                           - Arg Val Cys Trp Asn Gly Gln Lys Gly Gly Le - #u Glu Gly Leu Arg             #               255                                                           - Gln Lys Gly Trp Ser Ile Val Asn Leu Leu Va - #l Ile Gln Arg Glu             #               270                                                           - Ala Lys Ile Arg Asn Thr Ala Val Lys Val Le - #u Ala Gln Gly Asp             #               285                                                           - Asn Gln Val Ile Cys Thr Gln Tyr Lys Thr Ly - #s Lys Thr Arg Ser             #               300                                                           - Glu Leu Glu Leu Arg Ala Val Leu His Gln Me - #t Ala Gly Asn Asn             #               315                                                           - Asn Lys Ile Met Glu Glu Ile Lys Arg Gly Th - #r Glu Lys Leu Gly             #               330                                                           - Leu Ile Ile Asn Asp Asp Glu Thr Met Gln Se - #r Ala Asp Tyr Leu             #               345                                                           - Asn Tyr Gly Lys Ile Pro Ile Phe Arg Gly Va - #l Ile Arg Gly Leu             #               360                                                           - Glu Thr Lys Arg Trp Ser Arg Val Thr Cys Va - #l Thr Asn Asp Gln             #               375                                                           - Ile Pro Thr Cys Ala Asn Leu Met Ser Ser Va - #l Ser Thr Asn Ala             #               390                                                           - Leu Thr Val Ala His Phe Ala Glu Asn Pro Il - #e Asn Ala Met Ile             #               405                                                           - Gln Tyr Asn Tyr Phe Gly Thr Phe Ala Arg Le - #u Leu Leu Phe Met             #               420                                                           - His Asp Pro Ala Ile Arg Gln Ser Leu Tyr Ly - #s Val Gln Glu Lys             #               435                                                           - Ile Pro Gly Leu His Thr Arg Thr Phe Lys Ty - #r Ala Met Leu Tyr             #               450                                                           - Leu Asp Pro Ser Ile Gly Gly Val Cys Gly Me - #t Ala Leu Ser Arg             #               465                                                           - Phe Leu Ile Arg Ala Phe Pro Asp Pro Val Th - #r Glu Ser Leu Ser             #               480                                                           - Phe Trp Lys                                                                         483                                                                   - (2) INFORMATION FOR SEQ ID NO:35:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 515 amino                                                         (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -    (iii) HYPOTHETICAL:   NO                                                 -     (iv) ANTI-SENSE:  NO                                                    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:35:                                - Asn Lys Lys Ile Phe Gln Arg Ser Ser Leu Ty - #r Asn His Lys Asp             #                 15                                                          - Trp Asp Gln Val Val Ile Leu Gln Ser Phe Gl - #n Ile Pro Lys Ser             #                30                                                           - Val Asn Leu Ala Thr Met Ile Lys Asp Lys Al - #a Ile Ser Met Thr             #                45                                                           - Arg Ser Glu Leu Ile Glu Ser Val Asn Thr Ly - #s Asn Ser Val Phe             #                60                                                           - Asp Ser Thr Lys Arg Arg Gly Ile Leu Lys Tr - #p Leu Asn Glu Gln             #                75                                                           - Ser Asp Lys Ile Tyr Asn Phe Leu Met Arg Il - #e Asp Asp Lys Gly             #                90                                                           - Leu Asp Glu Asp Asp Cys Ile Ile Gly Leu Ty - #r Pro Lys Glu Arg             #                105                                                          - Glu Met Lys Thr Lys Ala Arg Phe Phe Ser Le - #u Met Ser Tyr Lys             #               120                                                           - Leu Arg Met Tyr Val Thr Ser Thr Glu Glu Le - #u Leu Gly Lys Tyr             #               135                                                           - Val Leu Lys Tyr Phe Pro Met Ile Thr Met Se - #r Asp Asn Leu Leu             #               150                                                           - Ser Met Val Ile Arg Leu Phe Asp Met Thr Th - #r Leu Ile Gly Asp             #               165                                                           - Lys Gly Val Ala Val Thr Tyr Ser Met Asn Il - #e Asp Phe Ser Lys             #               180                                                           - Trp Asn Gln Asn Met Arg Glu Arg Thr Asn Al - #a Gly Ile Phe Asp             #               195                                                           - Asn Leu Asp Arg Ile Leu Gly Phe Arg Ser Le - #u Ile Ser Arg Thr             #               210                                                           - His Ser Ile Phe Lys Ala Cys Tyr Leu Tyr Le - #u Cys Ser Gly Glu             #               225                                                           - Tyr Val Pro Val Ile Ser Asn Asn Gln Leu Th - #r Ala Gln Ser Pro             #               240                                                           - Trp Ser Arg Thr Gly Asp Glu Ser Gly Lys Gl - #u Gly Leu Arg Gln             #               255                                                           - Lys Gly Trp Thr Ile Thr Thr Val Cys Asp Il - #e Leu Ser Leu Ala             #               270                                                           - Phe Lys Tyr Asn Ala Arg Ile Gln Leu Ile Gl - #y Gly Gly Asp Asn             #               285                                                           - Gln Val Leu Thr Val Thr Met Leu Pro Ser Gl - #u Ser Met Gln Ser             #               300                                                           - Gln Gly Arg Asp Ser Gln Leu Leu Lys Val Ar - #g Glu Arg Met Thr             #               315                                                           - Ser Phe Arg Asn Ala Leu Ala Lys Lys Met Va - #l Lys Arg Gly Leu             #               330                                                           - Pro Leu Lys Leu Glu Glu Thr Trp Ile Ser Hi - #s Asn Leu Leu Met             #               345                                                           - Tyr Asn Lys Ile Met Tyr Tyr Ser Gly Val Pr - #o Leu Arg Gly Arg             #               360                                                           - Leu Lys Val Ile Ser Arg Leu Phe Ser Asn Se - #r Asn Val Gly Val             #               375                                                           - Thr Ser Leu Gly Gly Ile Thr Ser Thr Leu Gl - #y Thr Gly Phe Gln             #               390                                                           - Ser Ile Ser Thr Lys Asp Tyr Thr Pro Thr Le - #u Ala Trp Leu Ile             #               405                                                           - Ser Arg Val Phe Thr Asp Ile Tyr Ile Ser Th - #r Tyr His Leu Leu             #               420                                                           - Asn Pro Ile Ser Gly Thr Gln Arg Leu Asp Ly - #s Gln Val Leu Met             #               435                                                           - Ser Arg Gly Asn Ile Arg Gln Gly Arg Asn Gl - #u Leu Gly Gly Glu             #               450                                                           - Thr Ser Val Pro Ile Ile Asn Lys Ile Arg As - #n His Ala Ala Leu             #               465                                                           - Ala Thr Asp His Thr Leu Asp Leu Asp Ser Le - #u Leu Ile Cys Val             #               480                                                           - Leu Tyr Tyr His Lys Ile Leu Gly Gly Pro Gl - #y Ile Gly Pro Pro             #               495                                                           - Thr Ala Tyr Val Met Lys Gly Phe Pro Asp Pr - #o Leu Ser Glu Gly             #               510                                                           - Leu Thr Phe Asn Tyr                                                                         515                                                           - (2) INFORMATION FOR SEQ ID NO:36:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 535 amino                                                         (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -    (iii) HYPOTHETICAL:   NO                                                 -     (iv) ANTI-SENSE:  NO                                                    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:36:                                - Glu Gly Leu Thr His Glu Gln Cys Val Asp As - #n Trp Lys Ser Phe             #                 15                                                          - Ala Gly Val Lys Phe Gly Cys Phe Met Pro Le - #u Ser Leu Asp Ser             #                30                                                           - Asp Leu Thr Met Tyr Leu Lys Asp Lys Ala Le - #u Ala Ala Leu Gln             #                45                                                           - Arg Glu Trp Asp Ser Val Tyr Pro Lys Glu Ph - #e Leu Arg Tyr Asp             #                60                                                           - Pro Pro Lys Gly Thr Gly Ser Arg Arg Leu Va - #l Asp Val Phe Leu             #                75                                                           - Asn Asp Ser Ser Phe Asp Pro Tyr Asp Val Il - #e Met Tyr Val Val             #                90                                                           - Ser Gly Ala Tyr Leu His Asp Pro Glu Phe As - #n Leu Ser Tyr Ser             #                105                                                          - Leu Gln Glu Lys Glu Ile Lys Glu Thr Gly Ar - #g Leu Phe Ala Lys             #               120                                                           - Met Thr Tyr Lys Met Arg Ala Cys Gln Val Il - #e Ala Glu Asn Leu             #               135                                                           - Ile Ser Asn Gly Ile Gly Lys Tyr Phe Lys As - #p Asn Gly Met Ala             #               150                                                           - Lys Asp Glu Gln Asp Leu Thr Lys Ala Leu Hi - #s Thr Leu Ala Val             #               165                                                           - Ser Gly Val Pro Lys Asp Leu Lys Glu Ser Hi - #s Arg Gly Gly Pro             #               180                                                           - Val Leu Lys Thr Tyr Ser Arg Ser Pro Val Hi - #s Thr Ser Thr Arg             #               195                                                           - Asn Val Arg Ala Ala Lys Gly Phe Ile Gly Ph - #e Pro Gln Val Ile             #               210                                                           - Arg Gln Asp Gln Asp Thr Asp His Pro Glu As - #n Met Glu Ala Tyr             #               225                                                           - Glu Thr Val Ser Ala Phe Ile Thr Thr Asp Le - #u Lys Lys Tyr Cys             #               240                                                           - Leu Asn Trp Arg Tyr Glu Thr Ile Ser Leu Ph - #e Ala Gln Arg Leu             #               255                                                           - Asn Glu Ile Tyr Gly Leu Pro Ser Phe Phe Gl - #n Trp Leu His Lys             #               270                                                           - Arg Leu Glu Thr Ser Val Leu Tyr Val Ser As - #p Pro His Cys Pro             #               285                                                           - Pro Asp Leu Asp Ala His Ile Pro Leu Tyr Ly - #s Val Pro Asn Asp             #               300                                                           - Gln Ile Phe Ile Lys Tyr Pro Met Gly Gly Il - #e Glu Gly Tyr Cys             #               315                                                           - Gln Lys Leu Trp Thr Ile Ser Thr Ile Pro Ty - #r Leu Tyr Leu Ala             #               330                                                           - Ala Tyr Glu Ser Gly Val Arg Ile Ala Ser Le - #u Val Gln Gly Asp             #               345                                                           - Asn Gln Thr Ile Ala Val Thr Lys Arg Val Pr - #o Ser Thr Trp Pro             #               360                                                           - Tyr Asn Leu Lys Lys Arg Glu Ala Ala Arg Va - #l Thr Arg Asp Tyr             #               375                                                           - Phe Val Ile Leu Arg Gln Arg Leu His Asp Il - #e Gly His His Leu             #               390                                                           - Lys Ala Asn Glu Thr Ile Val Ser Ser His Ph - #e Phe Val Tyr Ser             #               405                                                           - Lys Gly Ile Tyr Tyr Asp Gly Leu Leu Val Se - #r Gln Ser Leu Lys             #               420                                                           - Ser Ile Ala Arg Cys Val Phe Trp Ser Glu Th - #r Ile Val Asp Glu             #               435                                                           - Thr Arg Ala Ala Cys Ser Asn Ile Ala Thr Th - #r Met Ala Lys Ser             #               450                                                           - Ile Glu Arg Gly Tyr Asp Arg Tyr Leu Ala Ty - #r Ser Leu Asn Phe             #               465                                                           - Leu Lys Val Ile Gln Gln Ile Leu Ile Ser Le - #u Gly Phe Thr Ile             #               480                                                           - Asn Ser Thr Met Thr Arg Asp Val Val Ile Pr - #o Leu Leu Thr Asn             #               495                                                           - Asn Asp Leu Leu Ile Arg Met Ala Leu Leu Pr - #o Ala Pro Ile Gly             #               510                                                           - Gly Met Asn Tyr Leu Asn Met Ser Arg Leu Ph - #e Val Arg Asn Ile             #               525                                                           - Gly Asp Pro Val Thr Ser Ser Ile Ala Asp                                     #               535                                                           - (2) INFORMATION FOR SEQ ID NO:37:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 527 amino                                                         (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -    (iii) HYPOTHETICAL:   NO                                                 -     (iv) ANTI-SENSE:  NO                                                    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:37:                                - Thr Ala Ile Ser Tyr Glu Cys Ala Val Asp As - #n Tyr Thr Ser Phe             #                 15                                                          - Ile Gly Phe Lys Phe Arg Lys Phe Ile Glu Pr - #o Gln Leu Asp Glu             #                30                                                           - Asp Leu Thr Ile Tyr Met Lys Asp Lys Ala Le - #u Ser Pro Arg Lys             #                45                                                           - Glu Ala Trp Asp Ser Val Tyr Pro Asp Ser As - #n Leu Tyr Tyr Lys             #                60                                                           - Ala Pro Glu Ser Glu Glu Thr Arg Arg Leu Il - #e Glu Val Phe Ile             #                75                                                           - Asn Asp Glu Asn Phe Asn Pro Glu Glu Ile Il - #e Asn Tyr Val Glu             #                90                                                           - Ser Gly Asp Trp Leu Lys Asp Glu Glu Phe As - #n Ile Ser Tyr Ser             #                105                                                          - Leu Lys Glu Lys Glu Ile Lys Gln Glu Gly Ar - #g Leu Phe Ala Lys             #               120                                                           - Met Thr Tyr Lys Met Arg Ala Val Gln Val Le - #u Ala Glu Thr Leu             #               135                                                           - Leu Ala Lys Gly Ile Gly Glu Leu Phe Ser Gl - #u Asn Gly Met Val             #               150                                                           - Lys Gly Glu Ile Asp Leu Leu Lys Arg Leu Th - #r Thr Leu Ser Val             #               165                                                           - Ser Gly Val Pro Arg Thr Asp Ser Val Tyr As - #n Asn Ser Lys Ser             #               180                                                           - Ser Glu Lys Arg Asn Glu Gly Met Gly Asn Ly - #s Asn Ser Gly Gly             #               195                                                           - Tyr Trp Asp Glu Lys Lys Arg Ser Arg His Gl - #u Phe Lys Ala Thr             #               210                                                           - Asp Ser Ser Thr Asp Gly Tyr Glu Thr Leu Se - #r Cys Phe Leu Thr             #               225                                                           - Thr Asp Leu Lys Lys Tyr Cys Leu Asn Trp Ar - #g Phe Glu Ser Thr             #               240                                                           - Ala Leu Phe Gly Gln Arg Cys Asn Glu Ile Ph - #e Gly Phe Lys Thr             #               255                                                           - Phe Phe Asn Trp Met His Pro Val Leu Glu Ar - #g Cys Thr Ile Tyr             #               270                                                           - Val Gly Asp Pro Tyr Cys Pro Val Ala Asp Ar - #g Met His Arg Gln             #               285                                                           - Leu Gln Asp His Ala Asp Ser Gly Ile Phe Il - #e His Asn Pro Arg             #               300                                                           - Gly Gly Ile Glu Gly Tyr Cys Gln Lys Leu Tr - #p Thr Leu Ile Ser             #               315                                                           - Met Ser Ala Ile His Leu Ala Ala Val Arg Va - #l Gly Val Arg Val             #               330                                                           - Ser Ala Met Val Gln Gly Asp Asn Gln Ala Il - #e Ala Val Thr Ser             #               345                                                           - Arg Val Pro Val Ala Gln Thr Tyr Lys Gln Ly - #s Lys Asn His Val             #               360                                                           - Tyr Glu Glu Ile Thr Lys Tyr Phe Gly Ala Le - #u Arg His Val Met             #               375                                                           - Phe Asp Val Gly His Glu Leu Lys Leu Asn Gl - #u Thr Ile Ile Ser             #               390                                                           - Ser Lys Met Phe Val Tyr Ser Lys Arg Ile Ty - #r Tyr Asp Gly Lys             #               405                                                           - Ile Leu Pro Gln Cys Leu Lys Ala Leu Thr Ly - #s Cys Val Phe Trp             #               420                                                           - Ser Glu Thr Leu Val Asp Glu Asn Arg Ser Al - #a Cys Ser Asn Ile             #               435                                                           - Ser Thr Ser Ile Ala Lys Ala Ile Glu Asn Gl - #y Tyr Ser Pro Ile             #               450                                                           - Leu Gly Tyr Cys Ile Ala Leu Tyr Lys Thr Cy - #s Gln Gln Val Cys             #               465                                                           - Ile Ser Leu Gly Met Thr Ile Asn Pro Thr Il - #e Ser Pro Thr Val             #               480                                                           - Arg Asp Gln Tyr Phe Lys Gly Lys Asn Trp Le - #u Arg Cys Ala Val             #               495                                                           - Leu Ile Pro Ala Asn Val Gly Gly Phe Asn Ty - #r Met Ser Thr Ser             #               510                                                           - Arg Cys Phe Val Arg Asn Ile Gly Asp Pro Al - #a Val Ala Ala Leu             #               525                                                           - Ala Asp                                                                         527                                                                       - (2) INFORMATION FOR SEQ ID NO:38:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 509 amino                                                         (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -    (iii) HYPOTHETICAL:   NO                                                 -     (iv) ANTI-SENSE:  NO                                                    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:38:                                - Ala Glu Ile Ser His Asp Ile Met Leu Arg Gl - #u Tyr Lys Ser Leu             #                 15                                                          - Ser Ala Leu Glu Phe Glu Pro Cys Ile Glu Ty - #r Asp Pro Val Thr             #                30                                                           - Asn Leu Ser Met Phe Leu Lys Asp Lys Ala Il - #e Ala His Pro Asn             #                45                                                           - Asp Asn Trp Leu Ala Ser Phe Arg Arg Asn Le - #u Leu Ser Glu Asp             #                60                                                           - Gln Lys Lys His Val Lys Glu Ala Thr Ser Th - #r Asn Arg Leu Leu             #                75                                                           - Ile Glu Phe Leu Glu Ser Asn Asp Phe Asp Pr - #o Tyr Lys Glu Met             #                90                                                           - Glu Tyr Leu Thr Thr Leu Glu Tyr Leu Arg As - #p Asp Asp Val Ala             #                105                                                          - Val Ser Tyr Ser Leu Lys Glu Lys Glu Val Ly - #s Val Asn Gly Arg             #               120                                                           - Ile Phe Ala Lys Leu Thr Lys Lys Leu Arg As - #n Cys Gln Val Met             #               135                                                           - Ala Glu Gly Ile Leu Ala Asp Gln Ile Ala Pr - #o Phe Phe Gln Gly             #               150                                                           - Asn Gly Val Ile Gln Asp Ser Ile Ser Leu Th - #r Lys Ser Thr Leu             #               165                                                           - Ala Met Ser Gln Leu Ser Phe Asn Ser Asn Ly - #s Lys Arg Ile Thr             #               180                                                           - Asp Cys Lys Glu Arg Val Ser Ser Asn Arg As - #n His Asp Pro Lys             #               195                                                           - Ser Lys Asn Arg Arg Arg Val Ala Thr Phe Il - #e Thr Thr Asp Leu             #               210                                                           - Gln Lys Tyr Cys Leu Asn Trp Arg Tyr Gln Th - #r Ile Lys Leu Phe             #               225                                                           - Ala His Ala Ile Asn Gln Leu Met Gly Leu Pr - #o His Phe Phe Glu             #               240                                                           - Trp Ile His Leu Arg Leu Met Asp Thr Thr Me - #t Phe Val Gly Asp             #               255                                                           - Pro Phe Asn Pro Pro Ser Asp Pro Thr Asp Cy - #s Asp Leu Ser Arg             #               270                                                           - Val Pro Asn Asp Asp Ile Tyr Ile Val Ser Al - #a Arg Gly Gly Ile             #               285                                                           - Glu Gly Leu Cys Gln Lys Leu Trp Thr Met Il - #e Ser Ile Ala Ala             #               300                                                           - Ile Gln Leu Ala Ala Ala Arg Ser His Cys Ar - #g Val Ala Cys Met             #               315                                                           - Val Gln Gly Asp Asn Gln Val Ile Ala Val Th - #r Arg Glu Val Arg             #               330                                                           - Ser Asp Asp Ser Pro Glu Met Val Leu Thr Gl - #n Leu His Gln Ala             #               345                                                           - Ser Asp Asn Phe Phe Lys Glu Leu Ile His Va - #l Asn His Leu Ile             #               360                                                           - Gly His Asn Leu Lys Asp Arg Glu Thr Ile Ar - #g Ser Asp Thr Phe             #               375                                                           - Phe Ile Tyr Ser Lys Arg Ile Phe Lys Asp Gl - #y Ala Ile Leu Ser             #               390                                                           - Gln Val Leu Lys Asn Ser Ser Lys Leu Val Me - #t Val Ser Gly Asp             #               405                                                           - Leu Ser Glu Asn Thr Val Met Ser Cys Ala As - #n Ile Ala Ser Thr             #               420                                                           - Val Ala Arg Leu Cys Glu Asn Gly Leu Pro Ly - #s Asp Phe Cys Tyr             #               435                                                           - Tyr Leu Asn Tyr Ile Met Ser Cys Val Gln Th - #r Tyr Phe Asp Ser             #               450                                                           - Glu Phe Ser Tyr Asn Asn Asn Ser His Pro As - #p Leu Asn Gln Ser             #               465                                                           - Trp Ile Glu Asp Ile Ser Phe Val His Ser Ty - #r Val Leu Thr Pro             #               480                                                           - Ala Gln Leu Gly Gly Leu Ser Asn Leu Gln Ty - #r Ser Arg Leu Tyr             #               495                                                           - Thr Arg Asn Ile Gly Asp Pro Gly Thr Thr Al - #a Phe Ala Glu                 #           509 505                                                           - (2) INFORMATION FOR SEQ ID NO:39:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 489 amino                                                         (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -    (iii) HYPOTHETICAL:   NO                                                 -     (iv) ANTI-SENSE:  NO                                                    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:39:                                - Ser Phe Pro Ser Gln Ala Glu Ile Tyr Gln Hi - #s Leu Trp Glu Trp             #                 15                                                          - Tyr Phe Val Glu His Glu Pro Leu Phe Ser Th - #r Lys Ile Ile Ser             #                30                                                           - Asp Leu Ser Ile Phe Ile Lys Asp Arg Leu Th - #r Ala Val Asn Gln             #                45                                                           - Glu Cys Trp Asp Ser Val Phe Asp Arg Ser Va - #l Leu Gly Tyr Asn             #                60                                                           - Pro Pro Val Arg Phe Gln Ser Lys Arg Val Pr - #o Glu Gln Phe Leu             #                75                                                           - Gly Gln Ala Asp Phe Ser Leu Asn Gln Ile Le - #u Glu Phe Ala Glu             #                90                                                           - Lys Leu Glu Tyr Leu Ala Pro Ser Tyr Arg As - #n Phe Ser Phe Ser             #                105                                                          - Leu Lys Glu Lys Glu Leu Asn Ile Gly Arg Th - #r Phe Gly Lys Leu             #               120                                                           - Pro Tyr Arg Val Arg Asn Val Gln Thr Leu Al - #a Glu Ala Leu Leu             #               135                                                           - Ala Asp Gly Leu Ala Lys Ala Phe Pro Ser As - #n Met Met Val Val             #               150                                                           - Thr Glu Arg Glu Gln Lys Glu Ala Leu Leu Hi - #s Gln Ala Ser Trp             #               165                                                           - His His Asn Ser Ala Ser Ile Gly Glu Asn Al - #a Ile Val Arg Gly             #               180                                                           - Ala Ser Phe Val Thr Asp Leu Glu Lys Tyr As - #n Leu Ala Phe Arg             #               195                                                           - Tyr Glu Phe Thr Arg His Phe Ile Asp Tyr Cy - #s Asn Arg Cys Tyr             #               210                                                           - Gly Val Lys Asn Leu Phe Asp Trp Met His Ph - #e Leu Ile Pro Leu             #               225                                                           - Cys Tyr Met His Val Ser Asp Phe Tyr Ser Pr - #o Pro His Cys Val             #               240                                                           - Thr Glu Asp Asn Arg Asn Asn Pro Pro Asp Cy - #s Ala Asn Ala Tyr             #               255                                                           - His Tyr His Leu Gly Gly Ile Glu Gly Leu Gl - #n Gln Lys Leu Trp             #               270                                                           - Thr Cys Ile Ser Cys Ala Gln Ile Thr Leu Va - #l Glu Leu Lys Thr             #               285                                                           - Lys Leu Lys Leu Lys Ser Ser Val Met Gly As - #p Asn Gln Cys Ile             #               300                                                           - Thr Thr Leu Ser Leu Phe Pro Ile Asp Ala Pr - #o Asn Asp Tyr Gln             #               315                                                           - Glu Asn Glu Ala Glu Leu Asn Ala Ala Arg Va - #l Ala Val Glu Leu             #               330                                                           - Ala Ile Thr Thr Gly Tyr Ser Gly Ile Phe Le - #u Lys Pro Glu Glu             #               345                                                           - Thr Phe Val His Ser Gly Phe Ile Tyr Phe Gl - #y Lys Lys Gln Tyr             #               360                                                           - Leu Asn Gly Val Gln Leu Pro Gln Ser Leu Ly - #s Thr Met Ala Arg             #               375                                                           - Cys Gly Pro Leu Ser Asp Ser Ile Phe Asp As - #p Leu Gln Gly Ser             #               390                                                           - Leu Ala Ser Ile Gly Thr Ser Phe Glu Arg Gl - #y Thr Ser Glu Thr             #               405                                                           - Arg His Ile Phe Pro Ser Arg Trp Ile Ala Se - #r Phe His Ser Met             #               420                                                           - Leu Ala Ile Asn Leu Leu Asn Gln Asn His Le - #u Gly Phe Pro Leu             #               435                                                           - Gly Phe Asn Ile Asp Ile Ser Cys Phe Lys Ly - #s Pro Leu Thr Phe             #               450                                                           - Ser Glu Lys Leu Ile Ala Leu Ile Thr Pro Gl - #n Val Leu Gly Gly             #               465                                                           - Leu Ser Phe Leu Asn Pro Glu Lys Leu Phe Ty - #r Arg Asn Ile Ser             #               480                                                           - Asp Pro Leu Thr Ser Gly Leu Phe Gln                                         #           489 485                                                           - (2) INFORMATION FOR SEQ ID NO:40:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 513 amino                                                         (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -    (iii) HYPOTHETICAL:   NO                                                 -     (iv) ANTI-SENSE:  NO                                                    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:40:                                - Thr Tyr Pro Ser Leu Leu Glu Leu Thr Glu Ar - #g Asp Leu Ile Val             #                 15                                                          - Leu Ser Gly Leu Arg Phe Tyr Arg Glu Phe Ar - #g Leu Pro Lys Lys             #                30                                                           - Val Asp Leu Glu Met Ile Ile Asn Asp Lys Al - #a Ile Ser Pro Pro             #                45                                                           - Lys Asn Leu Ile Trp Thr Ser Phe Pro Arg As - #n Tyr Met Pro Ser             #                60                                                           - His Ile Gln Asn Tyr Ile Glu His Glu Lys Le - #u Lys Phe Ser Glu             #                75                                                           - Ser Asp Lys Ser Arg Arg Val Leu Glu Tyr Ty - #r Leu Arg Asp Asn             #                90                                                           - Lys Phe Asn Glu Cys Asp Leu Tyr Asn Cys Va - #l Val Asn Gln Ser             #                105                                                          - Tyr Leu Asn Asn Pro Asn His Val Val Ser Le - #u Thr Gly Lys Glu             #               120                                                           - Arg Glu Leu Ser Val Gly Arg Met Phe Ala Me - #t Gln Pro Gly Met             #               135                                                           - Phe Arg Gln Val Gln Ile Leu Ala Glu Lys Me - #t Ile Ala Glu Asn             #               150                                                           - Ile Leu Gln Phe Phe Pro Glu Ser Leu Thr Ar - #g Tyr Gly Asp Leu             #               165                                                           - Glu Leu Gln Lys Ile Leu Glu Leu Lys Ala Gl - #y Ile Ser Asn Lys             #               180                                                           - Ser Asn Arg Tyr Asn Asp Asn Tyr Asn Asn Ty - #r Ile Ser Lys Cys             #               195                                                           - Ser Ile Ile Thr Asp Leu Ser Lys Phe Asn Gl - #n Ala Phe Arg Tyr             #               210                                                           - Glu Thr Ser Cys Ile Cys Ser Asp Val Leu As - #p Glu Leu His Gly             #               225                                                           - Val Gln Ser Leu Phe Ser Trp Leu His Leu Th - #r Ile Pro His Val             #               240                                                           - Thr Ile Ile Cys Thr Tyr Arg His Ala Pro Pr - #o Tyr Ile Gly Asp             #               255                                                           - His Ile Val Asp Leu Asn Asn Val Asp Glu Gl - #n Ser Gly Leu Tyr             #               270                                                           - Arg Tyr His Met Gly Gly Ile Glu Gly Trp Cy - #s Gln Lys Leu Trp             #               285                                                           - Thr Ile Glu Ala Ile Ser Leu Leu Asp Leu Il - #e Ser Leu Lys Gly             #               300                                                           - Lys Phe Ser Ile Thr Ala Leu Ile Asn Gly As - #p Asn Gln Ser Ile             #               315                                                           - Asp Ile Ser Lys Pro Ile Arg Leu Met Glu Gl - #y Gln Thr His Ala             #               330                                                           - Gln Ala Asp Tyr Leu Leu Ala Leu Asn Ser Le - #u Lys Leu Leu Tyr             #               345                                                           - Lys Glu Tyr Ala Gly Ile Gly His Lys Leu Ly - #s Gly Thr Glu Thr             #               360                                                           - Tyr Ile Ser Arg Asp Met Gln Phe Met Ser Ly - #s Thr Ile Gln His             #               375                                                           - Asn Gly Val Tyr Tyr Pro Ala Ser Ile Lys Ly - #s Val Leu Arg Val             #               390                                                           - Gly Pro Trp Ile Asn Thr Ile Leu Asp Asp Ph - #e Lys Val Ser Leu             #               405                                                           - Glu Ser Ile Gly Ser Leu Thr Gln Glu Leu Gl - #u Tyr Arg Gly Glu             #               420                                                           - Ser Leu Leu Cys Ser Leu Ile Phe Arg Asn Va - #l Trp Leu Tyr Asn             #               435                                                           - Gln Ile Ala Leu Gln Leu Lys Asn His Ala Le - #u Cys Asn Asn Lys             #               450                                                           - Leu Tyr Leu Asp Ile Leu Lys Val Leu Lys Hi - #s Leu Lys Thr Phe             #               465                                                           - Phe Asn Leu Asp Asn Ile Asp Thr Ala Leu Th - #r Leu Tyr Met Asn             #               480                                                           - Leu Pro Met Leu Phe Gly Gly Gly Asp Pro As - #n Leu Leu Tyr Arg             #               495                                                           - Ser Phe Tyr Arg Arg Thr Pro Asp Phe Leu Th - #r Glu Ala Ile Val             #               510                                                           - His Ser Val                                                                         513                                                                   - (2) INFORMATION FOR SEQ ID NO:41:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 43 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: genomic RNA                                         -    (iii) HYPOTHETICAL: NO                                                   -     (iv) ANTI-SENSE: NO                                                     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:41:                                # 43               AAGU UUGUUGUACG CAUUUUUUCG CGU                             - (2) INFORMATION FOR SEQ ID NO:42:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 54 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: genomic RNA                                         -    (iii) HYPOTHETICAL: NO                                                   -     (iv) ANTI-SENSE: NO                                                     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:42:                                - UAGUUAUUCG CACACAAAAG AUCCUAAAAA UUCUUCUUUC UUUUUGUGUG CC - #CA               54                                                                          - (2) INFORMATION FOR SEQ ID NO:43:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 46 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: genomic RNA                                         -    (iii) HYPOTHETICAL: NO                                                   -     (iv) ANTI-SENSE: NO                                                     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:43:                                #                 46AAA UCAUCAUCUC UUGUUUUUGU GUGUCU                          - (2) INFORMATION FOR SEQ ID NO:44:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 46 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: genomic RNA                                         -    (iii) HYPOTHETICAL: NO                                                   -     (iv) ANTI-SENSE: NO                                                     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:44:                                #                 46UCC CAUACAUGUU UUUUCUCUUG UUUGGU                          - (2) INFORMATION FOR SEQ ID NO:45:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 44 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: genomic RNA                                         -    (iii) HYPOTHETICAL: NO                                                   -     (iv) ANTI-SENSE: NO                                                     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:45:                                # 44               AUCG UAACUUACGG AUUCUCUGUU UGGU                            - (2) INFORMATION FOR SEQ ID NO:46:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 41 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: genomic RNA                                         -    (iii) HYPOTHETICAL: NO                                                   -     (iv) ANTI-SENSE: NO                                                     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:46:                                #   41             CUAU CCUUACCCAA CUUUGUUUGG U                               - (2) INFORMATION FOR SEQ ID NO:47:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 53 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: genomic RNA                                         -    (iii) HYPOTHETICAL: NO                                                   -     (iv) ANTI-SENSE: NO                                                     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:47:                                - UUUGCUUUGC AAUUGACAAU GUCUGUUUUU UCUUUGAUCU GGUUGUUAAG CG - #U                53                                                                          - (2) INFORMATION FOR SEQ ID NO:48:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 46 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: genomic RNA                                         -    (iii) HYPOTHETICAL: NO                                                   -     (iv) ANTI-SENSE: NO                                                     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:48:                                #                 46AAU UGUAAGAAUG GUUUUUUUGU CUUCGU                          - (2) INFORMATION FOR SEQ ID NO:49:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 48 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: genomic RNA                                         -    (iii) HYPOTHETICAL: NO                                                   -     (iv) ANTI-SENSE: NO                                                     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:49:                                #                48UAUG CAAGUUUGUU GUACGCAUUU UUUCGCGU                        - (2) INFORMATION FOR SEQ ID NO:50:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 58 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: genomic RNA                                         -    (iii) HYPOTHETICAL: NO                                                   -     (iv) ANTI-SENSE: NO                                                     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:50:                                - ACGAGAAAAA AAGUGUCAAA AACUAAUAUC UCGUAAUUUA GUUAAUUUUU UA - #AUAACU           58                                                                          - (2) INFORMATION FOR SEQ ID NO:51:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 50 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: genomic RNA                                         -    (iii) HYPOTHETICAL: NO                                                   -     (iv) ANTI-SENSE: NO                                                     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:51:                                #              50AUACAC AAAAUCAUCA UCUCUUGUUU UUGUGUGUCU                      - (2) INFORMATION FOR SEQ ID NO:52:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 60 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: genomic RNA                                         -    (iii) HYPOTHETICAL: NO                                                   -     (iv) ANTI-SENSE: NO                                                     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:52:                                - ACACACACAA AAAAGAUGAA GAAUGUUUUG UUUUACUUAU AUCAAAGCUU UU - #UUCUUAAU         60                                                                          - (2) INFORMATION FOR SEQ ID NO:53:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 59 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: genomic RNA                                         -    (iii) HYPOTHETICAL: NO                                                   -     (iv) ANTI-SENSE: NO                                                     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:53:                                - CCCUAAAAUC CUGUAUAACU UCAUUACAUA UCCCAUACAU GUUUUUUCUC UU - #GUUUGGU          59                                                                          - (2) INFORMATION FOR SEQ ID NO:54:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 65 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: genomic RNA                                         -    (iii) HYPOTHETICAL: NO                                                   -     (iv) ANTI-SENSE: NO                                                     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:54:                                - ACCAGACAAG AGUUUAAGAG AUAUGUAUCC UUUUAAAUUU UCUUGUCUUC UU - #GUAAGUUU         60                                                                          #            65                                                               - (2) INFORMATION FOR SEQ ID NO:55:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 62 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: genomic RNA                                         -    (iii) HYPOTHETICAL: NO                                                   -     (iv) ANTI-SENSE: NO                                                     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:55:                                - UGUUACAUUU UUGCUUUGCA AUUGACAAUG UCUGUUUUUU CUUUGAUCUG GU - #UGUUAAGC         60                                                                          #              62                                                             - (2) INFORMATION FOR SEQ ID NO:56:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 63 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: genomic RNA                                         -    (iii) HYPOTHETICAL: NO                                                   -     (iv) ANTI-SENSE: NO                                                     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:56:                                - ACGCUUAACA AAUAAACAAC AAAAAUGAGA AAAACAAUCA AACAACCAAA GG - #UUCAGAUU         60                                                                          #             63                                                              - (2) INFORMATION FOR SEQ ID NO:57:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 15 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: genomic RNA                                         -    (iii) HYPOTHETICAL: NO                                                   -     (iv) ANTI-SENSE: NO                                                     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:57:                                #    15                                                                       - (2) INFORMATION FOR SEQ ID NO:58:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 2658 base                                                         (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA to genomic RN - #A                             -    (iii) HYPOTHETICAL: NO                                                   -     (iv) ANTI-SENSE: NO                                                     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:58:                                #              50TGAAGG ACCTCAGGAA GAACCCCTCC ATGATCTCAG                      #             100ACCGGA AGGGAGCAGC TATCGAATGA TGAGCTTATC                      #             150GGAGCT GGCCGAGAAT AGCATGATCG AGGCTGAGGA                      #             200TTGGGG ACATCTCGGC TCGCATCGAG GCAGGGTTTG                      #             250CTCCAA GTGGAAACCA TCCAGACAGC TCAGCGGTGC                      #             300CATCAG AATCCTTGGC GAGAACATCA AGATACTGGA                      #             350CAATGA TGGAGACAAT GAAGCTCATG ATGGAGAAGG                      #             400GCATCA ACCGCCGTTG GGACCTCTGC ACCCATGTTG                      #             450ACCTCC GCGCATTTAT CCCCAGCTCC CAAGTGCCCC                      #             500GGGACA TCATACCATA AAAAAATCGA ATCACCATGA                      #             550TATGTG GAGCTCAAGG ACAAGGTAAT CGTCCCTGGA                      #             600GCTTGA GATAGACTTT GTAGGAGGGA CTTCACGGAA                      #             650TCCCAT TTCTTTCAGT GAAAGAGCCT CTGCAGCTTC                      #             700TTGACC GACTACTTCA CCATTGACGT AGAGCCAGCA                      #             750CAACAT ATACTTCCAG ATTGACGACT TCTTGCTCCT                      #             800TGTCCG TATACAAGGA CCCGATTAGG AAATACATGT                      #             850AAGGAA CAGAGCAAGC ACGCAATTAA TGCAGCTTTC                      #             900TCGGCT TCGGAACATT GGTGTTGGCC CTCTCGGCCC                      #             950CAGGGC CTTAGTTGCA ATACTGACTC CACTCCTGGA                      #            1000GATAAG GCGACTTTGC CACACCCCAA CGGAAAATGT                      #            1050TTAGTT ATCTTAACCA CACGACTATT AGCCTCCCGG                      #            1100TGCCTC AAGTACCACT GCAAAACCTA TTGGGGATTC                      #            1150CGCTGA CCGAATCATC AATCGGTACA CTGGTACTGT                      #            1200ACAACT CAGCGCCAGA GGATCCCTTC GAGTGCAACT                      #            1250TCGGCG ATTACAACAG AGATCTGCCG ATGCTCTATT                      #            1300GGCTGT ACAGACATTC CCACCGTTCA TGTACTGCAG                      #            1350GTACTG TGAGTCAGCA GGAGCTAGAG AGTGGAAAGG                      #            1400GGCAGT ACCTTAACTT ATACCCCGTA TATCTTACAA                      #            1450CAAAAC CCTTAATGGG ACTATACTCT GCAACTCATC                      #            1500CCTTCG ATGAATTTAG GCGTTCATAC TCCCTAGCGA                      #            1550AGCTCA TCAATCAATG TGACGTGTGT AAACTACACG                      #            1600CAAGTT GAGAAGGCGG CGTAGGGATA CTCAACAGAT                      #            1650ACAAGC TTAGGCCTAC ACTGAAAGAT GCGTGGGAGG                      #            1700CAGTCT CTGCTCCTAG GGGTGTTTGG TACTGGGATT                      #            1750ATTCTT GAGGGGCTGG CTCAACCACC CTGATATCAT                      #            1800ATGGAG TTGGGGTAGT CTGGCAATGC CATCGTGTTG                      #            1850GCGTGG AATGAGTCCA CATATTACCC TCCAGTAGAT                      #            1900GTACTT TCTGAATGAT GAGGGGAGGC TACAAACAAA                      #            1950GGCCAG GGCTTAAGCG GGTCATGTGG TTCGGCAGGT                      #            2000GTAGGG TCTGGGGTGA AACCGAGGAG GATTCGGTAC                      #            2050TGATTA CCATCTAGAG GAGTTTGAGG CAAGTCTCAA                      #            2100CCAGTA TCGCCTCGGG TCATGAGACA GACCCCATAA                      #            2150ACGCAG GCTGACCTCC TTCCATACAC CAGGTCTAGT                      #            2200AGATAC AGGCTCAGGC TGGGTGCACA TCGGCCTACC                      #            2250TCAATC CTCTCGGGTG GCTTAGGGAC CTACTTGCGT                      #            2300GGTGGG GTTCTATACT TAATAAGTCT TTGTGTTTCC                      #            2350CGCGAG GAGGAGACGC CTCGGCCGGT GGCAGGAATA                      #            2400ATCTCT TAAAAACCCT CTTCTCGGGA CAGAGGTCTC                      #            2450CGAGTT CACTCCCCCA TCACGTACGA GCATTGGGCC                      #            2500AACCTG GCATCCTGTG ACTATTACTT GCTATTCCGC                      #            2550CCCTGA AGTATATCCC ATTGGTGTCT TAATAAGAGC                      #            2600TAACAG TTATAGTATC AGCTTGGAAG CTGGATCACA                      #            2650TACTCC TCTGTGAGAT ATGCACTCAC CAATCCCCGG                      #        2658                                                                 - (2) INFORMATION FOR SEQ ID NO:59:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 5 amino                                                           (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -    (iii) HYPOTHETICAL:   NO                                                 -     (iv) ANTI-SENSE:  NO                                                    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:59:                                - Ser Asn Ser Gly Ser                                                         1               5                                                             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We claim:
 1. An isolated immunoreactive fragment of p23 selected fromthe group consisting of:a) SEQ ID NOs 20, 22, 23, 24, 25, 26 or 27; b)amino acid position 1 to amino acid position 102 of SEQ ID NO. 4; c)amino acid position 37 to amino acid position 201 of SEQ ID NO. 4; d)amino acid position 102 to amino acid position 201 of SEQ ID NO. 4; e)amino acid position 154 to amino acid position 201 of SEQ ID NO. 4; andf) an 8-mer peptide comprising at least four contiguous amino acids ofSEQ ID NOs 20, 21, 22, 23, 24, 25, 26 or
 27. 2. A composition comprisingat least one immunoreactive fragment of p23 according to claim
 1. 3. Animmunoassay method for detecting, in a sample, antibodies capable ofbinding one or more proteins selected from the group consisting of: p23,recp23 and immunogenic fragments thereof, said method comprising thesteps of:a) contacting the sample with one or more fragments of a fulllength amino acid sequence of p23, wherein at least one of saidfragments is selected from the group consisting of:(i) amino acidposition 1 to amino acid position 102 of p23, (ii) amino acid position37 to amino acid position 201 of p23, (iii) amino acid position 102 toamino acid position 201 of p23, (iv) amino acid position 154 to aminoacid position 201 of p23, (v) SEQ ID Nos. 20, 21, 22, 23, 24, 25, 26 or27, and (vi) an 8-mer peptide containing at least four contiguous aminoacids of SEQ ID NOs 20, 21, 22, 23, 24, 25, 26 or 27; and b) determiningbinding of the antibodies to the fragments.
 4. The immunoassay method ofclaim 3, wherein the sample is a biological sample from a human or otheranimal and the method is used to diagnose infection by BDV, or todiagnose, monitor and manage schizophrenia.
 5. An immunoassay method fordetecting, in a test sample, antibodies immunoreactive with Bornadisease virus, comprising the steps of:(a) contacting the test sample toone or more proteins selected from the group consisting of:i) a proteincomprising an amino acid sequence as set forth in SEQ ID NO:4, and ii)substitutional variants of the foregoing that bind anti-p23 antibody,said variants having one or more amino acid substitutions, wherein theamino acid substitutions are conservative, wherein the proteins areprebound to a solid support without gel size fractionation; and (b)detecting the binding of the antibodies to one or more of the proteins.6. A method for selecting for a ligand capable of binding p23 saidmethod comprising the steps of:a) contacting the ligand with a fragmentof a full length amino acid sequence of p23, wherein said fragment isselected from the group consisting of;(i) amino acid position 1 to aminoacid position 102 of p23, (ii) amino acid position 37 to amino acidposition 201 of p23, (iii) amino acid position 102 to amino acidposition 201 of p23, and (iv) amino acid position 154 to amino acidposition 201 of p23; (v) SEQ ID Nos. 20, 21, 22, 23, 24, 25, 26 or 27;and (vi) an 8-mer peptide containing at least four contiguous aminoacids of SEQ ID NOs 20, 21, 22, 23, 24, 25, 26 or 27; and b) selectingfor the ligand which is bound to the fragment.
 7. The method of claim 6,wherein said ligand is a monospecific antiserum and said selection stepfurther comprises eluting the ligand bound to the polypeptide.
 8. Themethod of claim 6, wherein said ligand is a monoclonal antibody.
 9. Anassay panel comprising one or more isolated immunoreactive fragments ofp23, wherein at least one of said fragments is selected from the groupconsisting of:(i) amino acid position 1 to amino acid position 102 ofp23; (ii) amino acid position 37 to amino acid position 201 of p23;(iii) amino acid position 102 to amino acid position 201 of p23; (iv)amino acid position 154 to amino acid position 201 of p23; (v) SEQ IDNos. 20, 22, 23, 24, 25, 26 or 27; and (vi) an 8-mer peptide, saidpeptide containing at least four contiguous amino acids of SEQ ID Nos.20, 21, 22, 23, 24, 25, 26 or
 27. 10. A preparation of immunogenicpolypeptides, said preparation comprising at least one fragment of afull length amino acid sequence of p23, capable of provoking a cellularor humoral immune response in an organism administered the preparation,wherein said fragment is selected from the group consisting of:(i) aminoacid position 1 to amino acid position 102 of p23; (ii) amino acidposition 37 to amino acid position 201 of p23; (iii) amino acid position102 to amino acid position 201 of p23; (iv) amino acid position 154 toamino acid position 201 of p23; and (v) SEQ ID Nos. 20, 22, 23, 24, 25,26, or 27; and (vi) an 8-mer peptide, said peptide containing at leastfour contiguous amino acids from SEQ ID Nos. 20, 21, 22, 23, 24, 25, 26or
 27. 11. A method for eliciting an immune response in an animalagainst BDV infection or related pathogenesis, said method comprisingadministering to the animal a preparation according to claim
 10. 12. Akit for detecting a Borna Disease Virus infection in animals or a BornaDisease-related human virus infection in humans comprising:a solidsupport; and a preparation comprising a virus-encoded protein, whereinsaid virus-encoded protein is encoded by the DNA of SEQ ID NO:3 andwherein said virus encoded protein is bound to said solid supportwithout gel size fractionation.
 13. The kit of claim 12 wherein thesolid support is selected from the group consisting of microtiter wells,test tubes, beads, strips, membranes, and microparticles.
 14. Animmunoreactive fragment of p23, said fragment containing an amino acidsequence selected from the group consisting of amino acid position 1 toamino acid position 102 of p23, amino acid position 37 to amino acidposition 201 of p23, amino acid position 102 to amino acid position 201of p23, SEQ ID NO:22, and SEQ ID NO:24.
 15. A substitutional variant ofa p23 fragment, wherein:(A) the p23 fragment is selected from the groupconsisting of;(1) an immunoreactive fragment according to claim 1, and(2) SEQ ID NO:21, and (B) the substitutional variant;(1) contains one ormore amino acid substitutions, wherein the amino acid substitutions areconservative, and (2) binds antibody in samples from patients having aBDV infection or neuropsychiatric disease but does not bind anydetectible antibodies in samples from patients having no BDV infectionand no neuropsychiatric disease.
 16. A fusion protein, comprising:(A) ap23 polypeptide comprising an immunoreactive region that binds anti-p23antibody, wherein said immunoreactive region is at least eightcontiguous amino acid residues of a full-length amino acid sequence ofp23, and (B) a heterologous polypeptide fused to the p23 polypeptide.17. A fusion protein according to claim 16, wherein the full-lengthamino acid sequence of p23 is SEQ ID NO:4.
 18. A fusion proteinaccording to claim 16, wherein the immunoreactive region contains atleast four contiguous amino acids from SEQ ID Nos. 20, 21, 22, 23, 24,25, 26 or
 27. 19. A fusion protein according to claim 16, wherein theimmunoreactive region is selected from the group consisting of:(1) aamino acid position 1 to amino acid position 102 of SEQ ID NO. 4; (2)amino acid position 37 to amino acid position 201 of SEQ ID NO. 4; (3)amino acid position 102 to amino acid position 201 of SEQ ID NO. 4; and(4) amino acid position 154 to amino acid position 201 of SEQ ID NO. 4.